Hydrothermal Vent Tubeworm Riftia Pachyptila Research Articles

Polyclonal symbiont populations in hydrothermal vent tubeworms and the environment

Horizontally transmitted symbioses usually house multiple and variable symbiont genotypes that are acquired from a much more diverse environmental pool via partner choice mechanisms. However, in the deep-sea hydrothermal vent tubeworm Riftia pachyptila (Vestimentifera, Siboglinidae), it has been suggested that the Candidatus Endoriftia persephone symbiont is monoclonal. Here, we show with high-coverage metagenomics that adult R. pachyptila house a polyclonal symbiont population consisting of one dominant and several low-frequency variants. This dominance of one genotype is confirmed by multilocus gene sequencing of amplified housekeeping genes in a broad range of host individuals where three out of four loci ( atpA, uvrD and recA) revealed no genomic differences, while one locus ( gyrB) was more diverse in adults than in juveniles. We also analysed a metagenome of free-living Endoriftia and found that the free-living population showed greater sequence variability than the host-associated population. Most juveniles and adults shared a specific dominant genotype, while other genotypes can dominate in few individuals. We suggest that although generally permissive, partner choice is selective enough to restrict uptake of some genotypes present in the environment.

Comparison of proton-specific ATPase activities in plume and root tissues of two co-occurring hydrocarbon seep tubeworm species Lamellibrachia luymesi and Seepiophila jonesi

Lamellibrachia luymesi and Seepiophila jonesi are co-occurring species of vestimentiferan tubeworms found at hydrocarbon seepage sites on the upper Louisiana slope of the Gulf of Mexico. Like all vestimentiferans, they rely on internal sulfide-oxidizing symbiotic bacteria for nutrition. These symbionts produce hydrogen ions as a byproduct of sulfide oxidation, which the host tubeworm needs to eliminate to prevent acidosis. The hydrothermal vent tubeworm Riftia pachyptila uses a high activity of P- and V-type H+-ATPases located in its plume epithelium to excrete protons. Unlike R. pachyptila, the seep species grow a posterior root, which they can use in addition to their plumes as a nutrient exchange surface. In this study we measured the ATPase activities of plume and root tissues collected from L. luymesi and S. jonesi, and used a combination of inhibitors to determine the relative activities of P- and V-type H+-ATPases. We found that the total H+-ATPase activity of their plumes was approximately 14 μmol h−1 g−1 wet weight, and that of their roots was between 5 and 7 μmol h−1 g−1 wet weight. These activities were more than ten times lower than those measured in R. pachyptila. We suggest that seep tubeworms might use passive channels to eliminate protons across their roots, in addition to ATP-dependant proton pumps located in their plumes and roots. In addition, we found strong differences between the types of ATPase activities in the plumes of L. luymesi and S. jonesi. While the H+-ATPase activity of L. luymesi plumes is dominated by P-type ATPases, S. jonesi has an unusually high activity of V-type H+-ATPases. We suggest that S. jonesi relies on its high V-type H+-ATPase activity to drive carbon dioxide uptake across its plume surface. L. luymesi, on the other hand, might rely partially on bicarbonate uptake across its root.

Identification, sequencing, and localization of a new carbonic anhydrase transcript from the hydrothermal vent tubeworm Riftia pachyptila

The vestimentiferan annelid Riftia pachyptila forms dense populations at hydrothermal vents along the East Pacific Rise at a depth of 2600 m. It harbors CO(2)-assimilating sulfide-oxidizing bacteria that provide all of its nutrition. To find specific host transcripts that could be important for the functioning of this symbiosis, we used a subtractive suppression hybridization approach to identify plume- or trophosome-specific proteins. We demonstrated the existence of carbonic anhydrase transcripts, a protein endowed with an essential role in generating the influx of CO(2) required by the symbionts. One of the transcripts was previously known and sequenced. Our quantification analyses showed a higher expression of this transcript in the trophosome compared to the branchial plume or the body wall. A second transcript, with 69.7% nucleotide identity compared to the previous one, was almost only expressed in the branchial plume. Fluorescent in situ hybridization confirmed the coexpression of the two transcripts in the branchial plume in contrast with the trophosome where only one transcript could be detected. An alignment of these translated carbonic anhydrase cDNAs with vertebrate and nonvertebrate carbonic anhydrase protein sequences revealed the conservation of most amino acids involved in the catalytic site. According to the phylogenetic analyses, the two R. pachyptila transcripts clustered together but not all nonvertebrate sequences grouped together. Complete sequencing of the new carbonic anhydrase transcript revealed the existence of two slightly divergent isoforms probably coded by two different genes.

Toward a mechanistic understanding of larval dispersal: insights from genomic fingerprinting of the deep‐sea hydrothermal vent tubeworm Riftia pachyptila

AbstractLarval dispersal is critical for the maintenance of species populations in patchy and ephemeral hydrothermal vent habitats. On fast‐spreading ridges, such as the East Pacific Rise, rates of habitat turnover are comparable to estimated lifespans of many of the inhabiting species. Traditionally, dispersal questions have been addressed with two very different approaches, larval studies and population genetics. Population genetic studies of vent‐endemic species have been informative for determining whether patterns of dispersal are suggestive of stepping stone or island models and estimating rates of gene flow (effective migrants per generation) over broad geographic ranges. However, these studies leave fundamental questions unanswered about the specific mechanisms by which larvae disperse and species maintain their populations and biogeographic ranges. With the goal of examining genetic structure and elucidating alternative larval dispersal mechanisms, we employed a genomic DNA fingerprinting technique, amplified fragment length polymorphisms (AFLPs). To assess the potential utility of AFLPs, and genetic structure of the hydrothermal vent tubeworm Riftia pachyptila, genomic ‘fingerprints’ were recovered from 29 individuals from five vent fields spanning a distance of up to c. 5000 km along the East Pacific Rise. In contrast to previous population genetic studies that found little to no genetic structure using allozymes and mitochondrial DNA, genetic analyses of 630 polymorphic AFLP loci identified distinct subclades within R. pachyptila populations. Significant levels of differentiation were observed among populations from all vent regions as well as within each region. Discrete assemblages of tubeworms separated by as little as c. 400 m within a given vent region were genetically distinguishable and cohorts (based on size‐frequency distribution) within an aggregation were found to be most closely related. These results suggest that mechanisms of larval dispersal act to retain cohort fidelity in R. pachyptila.

Metabolite uptake, stoichiometry and chemoautotrophic function of the hydrothermal vent tubeworm Riftia pachyptila: responses to environmental variations in substrate concentrations and temperature

The hydrothermal vent tubeworm Riftia pachyptila is a dominant member of many hydrothermal vent communities along the East Pacific rise and is one of the fastest growing metazoans known. Riftia flourish in diffuse hydrothermal fluid flows, an environment with high spatial and temporal heterogeneity in physical and chemical conditions. To date, physiological and biochemical studies of Riftia have focused on Riftia's adaptations to its chemoautotrophic bacterial symbionts. However the relation between in situ physico-chemical heterogeneity and Riftia host and symbiont metabolism, in particular symbiont chemoautotrophic function, remain poorly understood. Accordingly, we conducted experiments using shipboard high-pressure respirometers to ascertain the effect of varying substrate concentrations and temperature on Riftia metabolite uptake and symbiont carbon fixation. Our results show that substrate concentrations can strongly govern Riftia oxygen and sulfide uptake rates, as well as net carbon uptake (which is a proxy for chemoautotrophic primary production). However, after sufficient exposure to sulfide and oxygen, Riftia were capable of sustaining symbiont autotrophic function for several hours in seawater devoid of sulfide or oxygen, enabling the association to support symbiont metabolism through brief periods of substrate deficiency. Overall, temperature had the largest influence on Riftia metabolite uptake and symbiont autotrophic metabolism. In sum, while Riftia requires sufficient availability of substrates to support symbiont chemoautotrophic function, it is extremely well poised to buffer the temporal and spatial heterogeneity in environmental substrate concentrations, alleviating the influence of environmental heterogeneity on symbiont chemoautotrophic function.

Experimental application of vascular and coelomic catheterization to identify vascular transport mechanisms for inorganic carbon in the vent tubeworm, Riftia pachyptila

Maintaining deep sea animals in in situ conditions has always been technically difficult because of the high-pressure requirements. Even more difficult are any attempts in manipulating or sampling these organisms while keeping them alive in high-pressure aquaria. We present a technique to withdraw blood samples by vascular catheterization which allows withdrawal of samples of during maintenance of specimens under high-pressure conditions. We have developed this technique to answer a long debated question, how carbon dioxide is transported from the ambient sea water to the bacterial symbionts inside the trophosome of the hydrothermal vent tubeworm Riftia pachyptila. Our results indicate that the carbon supply to the symbionts is mainly through inorganic CO 2 while its incorporation into malate and succinate may serve storage functions at periods of low CO 2 availability in the environment.

Characterization of carbonic anhydrases from Riftia pachyptila, a symbiotic invertebrate from deep-sea hydrothermal vents.

The symbiotic hydrothermal vent tubeworm Riftia pachyptila needs to supply its internal bacterial symbionts with carbon dioxide, their inorganic carbon source. Our aim in this study was to characterize the carbonic anhydrase (CA) involved in CO(2) transport and conversion at various steps in the plume and the symbiotic tissue, the trophosome. A complete 1209 kb cDNA has been sequenced from the trophosome and identified as a putative alpha-CA based on BLAST analysis and the similarities of total deduced amino-acid sequence with those from the GenBank database. In the plume, the putative CA sequence obtained from cDNA library screening was 90% identical to the trophosome CA, except in the first 77 nucleotides downstream from the initiation site identified on trophosome CA. A phylogenetic analysis showed that the annelidan Riftia CA (CARp) emerges clustered with invertebrate CAs, the arthropodan Drosophila CA and the cnidarian Anthopleura CA. This invertebrate cluster appeared as a sister group of the cluster comprising mitochondrial and cytosolic isoforms in vertebrates: CAV, CAI II and III, and CAVII. However, amino acid sequence alignment showed that Riftia CA was closer to cytosolic CA than to mitochondrial CA. Combined biochemical approaches revealed two cytosolic CAs with different molecular weights and pI's in the plume and the trophosome, and the occurrence of a membrane-bound CA isoform in addition to the cytosolic one in the trophosome. The physiologic roles of cytosolic CA in both tissues and supplementary membrane-bound CA isoform in the trophosome in the optimization of CO(2) transport and conversion are discussed.

Effects of metabolite uptake on proton-equivalent elimination by two species of deep-sea vestimentiferan tubeworm, Riftia pachyptila and Lamellibrachia cf luymesi: proton elimination is a necessary adaptation to sulfide-oxidizing chemoautotrophic symbionts.

Intracellular symbiosis requires that the host satisfy the symbiont's metabolic requirements, including the elimination of waste products. The hydrothermal vent tubeworm Riftia pachyptila and the hydrocarbon seep worm Lamellibrachia cf luymesi are symbiotic with chemolithoautotrophic bacteria that produce sulfate and protons as end-products. In this report, we examine the relationship between symbiont metabolism and host proton equivalent elimination in R. pachyptila and L. cf luymesi, and the effects of sulfide exposure on proton-equivalent elimination by Urechis caupo, an echiuran worm that lacks intracellular symbionts (for brevity, we will hereafter refer to proton-equivalent elimination as 'proton elimination'). Proton elimination by R. pachyptila and L. cf luymesi constitutes the worms' largest mass-specific metabolite flux, and R. pachyptila proton elimination is, to our knowledge, the most rapid reported for any metazoan. Proton elimination rates by R. pachyptila and L. cf luymesi correlated primarily with the rate of sulfide oxidation. Prolonged exposure to low environmental oxygen concentrations completely inhibited the majority of proton elimination by R. pachyptila, demonstrating that proton elimination does not result primarily from anaerobic metabolism. Large and rapid increases in environmental inorganic carbon concentrations led to short-lived proton elimination by R. pachyptila, as a result of the equilibration between internal and external inorganic carbon pools. U. caupo consistently exhibited proton elimination rates 5-20 times lower than those of L. cf luymesi and R. pachyptila upon exposure to sulfide. Treatment with specific ATPase inhibitors completely inhibited a fraction of proton elimination and sulfide and inorganic carbon uptake by R. pachyptila, suggesting that proton elimination occurs in large part via K(+)/H(+)-ATPases and Na(+)/H(+)-ATPases. In the light of these results, we suggest that protons are the primary waste product of the symbioses of R. pachyptila and L. cf luymesi, and that proton elimination is driven by symbiont metabolism, and may be the largest energetic cost incurred by the worms.

Biometry of the branchial plume in the hydrothermal vent tubewormRiftia pachyptila(Vestimentifera; Annelida)

The branchial plume of the hydrothermal vent tubeworm Riftia pachyptila is the main organ by which this mouth- and gut-less tubeworm directly exchanges metabolites with its environment. We estimated the total branchial surface area per unit wet mass, termed the specific branchial surface area (SBSA), from planimetric measurements. Changes in the SBSA during the growth of the worm were inferred from 16 individuals ranging from 1 to 112 g wet mass. Riftia pachyptila has a mean SBSA of 22 cm2·g–1, the second highest among all aquatic animals, representing 9 times the surface area of the rest of the body. Three significantly different classes of SBSA could be distinguished, corresponding to small, medium-sized, and large individuals. The SBSA values for small and medium-sized R. pachyptila are twice that for large individuals. Negative growth allometry between the length of the branchial plume and that of the trunk may be correlated with this variation in SBSA, the plume growing faster than the trunk in the small and medium-sized groups. In large individuals the trunk length exceeds the plume length, inducing an increase in body mass that lowers the SBSA. However, a lower SBSA does not imply reduced metabolite diffusion through the plume of large tubeworms, since their longer free filaments bear more developed pinnules, which are probably the preferred pathway of metabolite diffusion, owing to a minimal transepithelial distance of 2 µm.

Activity and inhibitor sensitivity of ATPases in the hydrothermal vent tubeworm Riftia pachyptila : a comparative approach

Phosphorylated ATPases may be involved in the effective pH regulation seen in the hydrothermal vent tubeworm Riftia pachyptila. R. pachyptila appears not only to have a large concentration of ATPases, but the main function of these ATPases seems to have shifted from other types of transport, such as Na+ and K+ movement, to the facilitation of H+ elimination. Plume and trophosome ATPase activity for R. pachyptila measured 646.2 ± 29.5 and 481.4 ± 32.0 μmol Pi (inorganic phosphate) g−1 wet wt h−1, respectively. Plume tissue ATPase activity (both mass-specific and protein-specific) in R. pachyptila was higher (between 7% and 55%) than the activity measured in any tissue for 7 other shallow- and deep-living species, in this study. This supports the hypothesis that R. pachyptila regulates acid/base balance via high concentrations of H+-ATPases, including Na+/H+ and K+/H+ exchangers and possibly electrogenic H+-ATPases, as evidenced by a higher total ATPase concentration (646 μmol Pi g−1 wet wt h−1), lesser Na+/K+-ATPase activity (13% of the total, as compared to 20−40% found in other animals), and higher H+-ATPase activity (226–264 μmol Pi g−1wet wt h−1). Overall, R. pachyptila appears to demonstrate elevated ATPase activity, with a greater fraction of the enzymes devoted to proton elimination, in order to effectively control its extracellular pH in the face of processes acting to acidify the internal environment.

Nitrogen metabolites and related enzymatic activities in the body fluids and tissues of the hydrothermal vent tubeworm Riftia pachyptila.

The distribution of nitrogen metabolism end-products and the associated enzyme activities, free amino acids and purine base catabolites were investigated in all the body compartments (circulating fluids and tissues) of the hydrothermal vent tubeworm Riftia pachyptila to acquire a general overview of nitrogen metabolism in this symbiotic organism. There were striking differences between the symbiont-containing trophosome tissue and other host tissues. High concentrations of ammonia, creatinine and, in particular, urate were found in all tissues, but they were present at consistently higher concentrations in the trophosome, which also contained large amounts of urea. Uric acid crystals were present at the periphery of trophosome lobules. The urea cycle appears to be fully functional in this tissue, which also uses creatine phosphate for phosphagen storage, while arginine phosphate or a combination of both phosphagens occurs in other tissues. The amino acid patterns are dominated by sulphated compounds in all tissues except the trophosome, which has high levels of aspartate and glutamate. Although no definitive conclusions could be drawn regarding the nitrogen regime of Riftia pachyptila, this in vitro study gives several indications for future research in this area.

Primary structure of the common polypeptide chainb from the multi-hemoglobin system of the hydrothermal vent tube wormRiftia pachyptila: An insight on the sulfide binding-site

The deep-sea tube worm Riftia pachyptila Jones possesses a multi-hemoglobin system with three different extracellular Hbs: two dissolved in the vascular blood, V1 (ca. 3,500 kDa) and V2 (ca. 400 kDa), and one in the coelomic fluid, C1 (ca. 400 kDa). V1 Hb consists of four heme-containing, globin chains (b-e) and four linker chains (L1-L4). V2 and C1 Hbs are exclusively built from globin chains, six for V2 (a-f) and five for C1 (a-e). The complete amino acid sequence of the isolated monomeric globin chain b, common to all Riftia Hbs, has been determined by automated Edman degradation sequencing of the peptides derived by digestion with trypsin, chymotrypsin, thermolysin, and CNBr. This polypeptide chain is composed of 144 amino acid residues, providing a M(r) of 16, 135.0 Da. Moreover, the primary sequence of chain b revealed 3 Cys residues at position 4, 75, and 134. Cys-4 and Cys-134 are located at positions where an intra-chain disulfide bridge is formed in all annelid, vestimentiferan, or pogonophoran chains, but Cys-75 is located at a unique position only found in three globin chains belonging to Lamellibrachia and Oligobrachia, a vestimentiferan and a pogonophoran. In both groups, Hbs can bind sulfide reversibly to fuel the chemosynthetic process of the symbiotic bacteria they harbor. Sulfide-binding experiments performed on purified Hb fractions (i.e., V1, V2, and C1 Hbs) suggest that free Cys residues on globin chains, and the numerous Cys found in linker chains, as determined previously by ESI-MS, may be the sulfide binding-sites. Blocking the free Cys by N-ethylmaleimide, we confirmed that free cysteines were involved in sulfide-binding but did not account for the whole sulfide-binding capacity of V1 Hb. Furthermore, a phylogenetic tree was constructed from 18 globin-like chains of annelid, vestimentiferan, and pogonophoran extracellular Hbs to clarify the systematic position of tubeworms. Riftia chain b clearly belongs to the "strain A" family with 30 to 80% identity with the other sequences analyzed. Its position in the tree confirmed a close relationship between vestimentiferan, pogonophoran, and annelid Hbs.

A histidine protein kinase homolog from the endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila.

The uncultivated bacterial endosymbionts of the hydrothermal vent tubeworm Riftia pachyptila play a central role in providing their host with fixed carbon. While this intimate association between host and symbiont indicates tight integration and coordination of function via cellular communication mechanisms, no such systems have been identified. To elucidate potential signal transduction pathways in symbionts that may mediate symbiont-host communication, we cloned and characterized a gene encoding a histidine protein kinase homolog isolated from a symbiont fosmid library. The gene, designated rssA (for Riftia symbiont signal kinase), resembles known sensor kinases and encodes a protein capable of phosphorylating response regulators in Escherichia coli. A second open reading frame, rssB (for Riftia symbiont signal regulator), encodes a protein similar to known response regulators. These results suggest that the symbionts utilize a phosphotransfer signal transduction mechanism to communicate external signals that may mediate recognition of or survival within the host. The specific signals eliciting a response by the signal transduction proteins of the symbiont remain to be elucidated.

Rate and process of tube production by the deep-sea hydrothermal vent tubeworm Riftia pachyptila

Rate and process of tube production by the deep-sea hydrothermal vent tubeworm Riftia pachyptila

Three-dimensional reconstruction of the hexagonal bilayer hemoglobin of the hydrothermal vent tube worm Riftia pachyptila by cryoelectron microscopy.

A frozen-hydrated specimen of the V1 hemoglobin of the hydrothermal vent tube worm Riftia pachyptila was observed in the electron microscope and subjected to three-dimensional reconstruction by the method of random conical tilt series. The 3D volume possesses a D6 point-group symmetry. When viewed along its 6-fold axis the vertices of its upper hexagonal layer are 16 degrees clockwise rotated compared to those of the lower layer. A central linker complex is decorated by 12 hollow globular substructures. The linker complex comprises (i) a central hexagonal toroid, (ii) two internal bracelets onto which the hollow globular substructures are built, and (iii) six structures connecting the two hexagonal layers. The hollow globular substructures, related to the dodecamers of globin chains resulting from the dissociation of the hexagonal bilayer hemoglobin, have a local pseudo 3-fold symmetry and are composed each of three elongated structures visible when the volume is displayed at high threshold. At a resolution of 36 A, the 3D volumes of the hexagonal bilayer hemoglobins of Riftia pachyptyla and of the leech Macrobdella decora look almost perfectly identical.

Unusual carbon dioxide-combining properties of body fluids in the hydrothermal vent tubeworm Riftia pachyptila

Total CO2 (ΣCO2) up to 50 mmol l−1, corresponding to internal CO2 partial pressures (PCO2) up to 6 kPa (1 kPa ≈ 7.5 mm Hg or Torr), have been measured in the body fluids of the hydrothermal vent tubeworm Riftia pachyptila (Jones) sampled at 13°N on the East Pacific Rise. At physiological pH values, such high ΣCO2 and PCO2 are quite unusual in water-breathing animals. In Riftia, both blood and coelomic fluid contain extracellular haemoglobins at fairly high concentrations, but their titration by a strong acid as well as CO2 equilibration experiments on dialysed and ultra-filtered fluids showed that the presence of these proteins account for only one-tenth to one-third of the observed ΣCO2. Gel filtration analysis of the fluids revealed that the protein-free fractions retained most of the CO2: about 64% in blood and 80% in coelomic fluid. This corresponds to a base excess of unknowm nature and origin at a concentration up to 30 meq l−1. The nutritional needs of the mouthless and gutless Riftia are totally derived from the metabolic activity of sulphide-oxidizing, chemolithoautotrophic bacterial symbionts able to fix inorganic carbon into organic molecules. In an external environment in which PCO2 varies widely due to the turbulent mixing of the hot, sulphide- and CO2-rich vebt water with the cold, CO2-poor deep sea water, we propose that the base excess has three main functions: first, to retain CO2 in the body fluids when the external PCO2 is low; second, to act as a buffer when external PCO2 is high; third, to allow the large coelomic compartment to act as a CO2 store, thus permitting the symbiotic bacteria to fix carbon continuously even when the external supply of carbon dioxide fluctuates.

The Sulphide-Binding Protein in the Blood of the Vestimentiferan Tube-Worm, Riftia Pachyptila, is the Extracellular Haemoglobin

ABSTRACT The sulphide-binding protein that occurs in high concentrations in the vascular blood and coelomic fluid of the hydrothermal vent tube-worm Riftia pachyptila Jones is the haemoglobin. Sulphide binding does not occur at the oxygen-binding sites of the haem, but may occur via thiol-disulphide exchange at the interchain disulphide bridges on the macromolecule. We have confirmed the report that vascular blood is heterogeneous for two haemoglobins (FI and FII) that are different in MT, but we conclude that the coelomic fluid is homogeneous for the lower Mr haemoglobin FII, in the intact, living animal. These two haemoglobins occur naturally in the living animals, and FII is not a dissociation product of the higher Mr FI. The sulphide-binding capacities of the two haemoglobin species differ by about a factor of two. Consequently, the vascular blood and the coelomic fluid also have different sulphide-binding capacities. These differences in sulphide-binding capacity may have important ramifications for the physiology of this unusual animal.

Metabolic and blood characteristics of the hydrothermal vent tube-worm Riftia pachyptila

Specimens of the hydrothermal vent pogonophoran Riftia pachyptila Jones were collected by submersible at a depth of 2 600 m at the 21°N hydrothermal vent site on the East Pacific Rise (20°50′N, 109°06′W) in April and May of 1982. The worms were maintained in pressurized aquaria for up to 45 d for metabolic studies. Consumption of O2 was regulated down to low PO2 (oxygen partial pressure) values; O2 consumption rates were 0.63 and 1.12 μ mol g-1 wet wt h-1 at 2.5° and 8°C, respectively; such rates were comparable to those previously measured for other pogonophorans. Intact specimens of R. pachyptila (including bacterial symbionts) did not consume significant amounts of CH4 from the environment. The respiratory quotients, in the absence of added sulfide, indicated that metabolism was mainly heterotrophic. High rates of uptake of dissolved amino acids were recorded for one specimen. The total [CO2] in the vascular blood and the Hb-containing coelomic fluid were high. Under anaerobic conditions, there were equilibrium distributions of pH, total [CO2] and sulfide concentrations between the vascular blood and the coelomic fluid, apparently because these metabolites were readily exchanged between the two compartments. The vascular blood bound neither CH4 nor H2. However, sulfide was reversibly bound by both the vascular blood and coelomic fluid; because this binding depended strongly on pH (with a maximum at about 7.5), HS- was probably the molecular species bound. Under anaerobic, but not aerobic conditions, the trophosome bound substantial amount of sulfide; thus, the high concentrations of sulfide in the trophosome may have resulted mainly from sulfide bound to sulfide oxidases under anaerobic conditions. The coelomic fluid had a relatively low buffering capacity (2.2 mmol CO2ΔpH-1).