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Coupling Metabolite Flux to Transcriptomics: Insights Into the Molecular Mechanisms Underlying Primary Productivity by the Hydrothermal Vent Tubeworm Ridgeia piscesae

¹ University of Connecticut, Department of Molecular and Cell Biology, 91 North Eagleville Road, Unit 3125, Storrs, Connecticut 06269-3125
² Harvard University, Organismic and Evolutionary Biology, 16 Divinity Avenue, Room 3085, Cambridge, Massachusetts 02138-2020

^* To whom correspondence should be addressed. E-mail: pgirguis{at}oeb.harvard.edu

	Abstract

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Deep-sea hydrothermal vents host highly productive ecosystems. Many of these communities are dominated by vestimentiferan tubeworms that house endosymbiotic chemoautotrophic bacteria that provide the hosts with their primary nutritional needs. Rates of carbon fixation by these symbioses are also among the highest recorded. Despite the breadth of physiological and biochemical research on these associations, the underlying molecular mechanisms that regulate host and symbiont metabolite flux and carbon fixation are largely unknown. Here we present metabolite flux and transcriptomics data from shipboard high-pressure respirometry experiments in which we maintained Ridgeia piscesae tubeworms at conditions comparable to those in situ. Host trophosome was used for cDNA library construction and sequencing. Of the 19,132 clones sequenced, 10,684 represented unique expressed sequence tags (ESTs). The highest proportions of genes are involved with translation, ribosomal structure and biogenesis, cellular processing, and signal transduction. There was moderate representation of genes involved in metabolite exchange and acid-base regulation. These data represent the first concomitant surveys of metabolite flux rates and gene expression for a chemoautotrophic symbiosis during net autotrophy, and they suggest that—in the case of Ridgeia piscesae—host-symbiont interactions such as cell cycle regulation may play a significant role in maintaining physiological poise during high productivity.

Abbreviations: EST, expressed sequence tag • HPRS, high-pressure respirometry system

	Introduction

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Thirty years ago, scientists discovered active hydrothermal vents in the ocean off the Galapagos Islands (Corliss et al., 1979). Like the findings of Darwin 140 years earlier, the discovery of hydrothermal vents on the Galapagos rift fundamentally altered our notions about life on Earth. These underwater hot springs discharge chemically altered seawater that is low in pH and enriched in hydrogen sulfide, iron, and other metals and gasses (Von Damm, 1990). Although temperatures of vent effluents can exceed 380 °C (Urabe et al., 1995; Schmidt et al., 2007), mixing with cold seawater results in biologically habitable regimes. Indeed, hydrothermal vents support extensive biological communities, comparable in biomass to rainforests and coral reefs (Gaill et al., 1997; Sarrazin and Juniper, 1999; Govenar et al., 2005). Although a majority of vent endemic species are heterotrophs, animals that have nutritional symbioses with chemoautotrophic bacteria constitute as much as 96% of the community biomass (Sarrazin and Juniper, 1999; Govenar et al., 2004). These chemoautotrophic symbionts harness energy via the oxidation of reduced chemical species and fix inorganic compounds for growth and biosynthesis. The co-availability of chemically reduced and oxidized compounds within vent mixing zones (called diffuse flows) is ideal for chemoautotrophic activity.

Hydrothermal vent tubeworms were the first chemoautotrophic symbioses to be described (Jones, 1981). The best known of these symbioses is the giant vent tubeworm Riftia pachyptila Jones, 1981 (hereafter referred to as Riftia), a monospecific genus within the family Siboglinidae (Rouse, 2001). Tubeworms are the dominant megafaunal species at many vent sites, growing in enormous aggregations and hosting numerous other species such as mussels, polychaete worms, limpets, and crabs (Hessler and Smithey, 1983; Shank et al., 1998; Govenar et al., 2005). Tubeworms are devoid of a mouth or digestive tract, and host their intracellular chemoautotrophic bacteria within a vascularized organ called the trophosome (Cavanaugh et al., 1981; Felbeck, 1981). These worms cannot ingest particulate organic matter; therefore they are entirely reliant on their symbionts for nutrition and must provide all the substrates necessary for symbiont chemoautotrophy. As a result, vent vestimentiferans thrive in diffuse flows, positioning their plumelike gill at the interface of vent flow and bottom-water mixing (Childress et al., 1991). Because these diffuse flows are spatially and temporally heterogeneous, with large fluctuations in chemistry and temperature (Johnson et al., 1986, 1988; Chevaldonne et al., 1991; Shank et al., 1998; Luther et al., 2001; Mullineaux et al., 2003; Le Bris et al., 2005), vent vestimentiferans have evolved to rapidly acquire and store substrates from the environment to dampen the variations in substrate availability. For example, they can bind both oxygen and sulfide in their blood (Arp and Childress, 1983; Flores et al., 2005), and Riftia has been shown to store enough oxygen and sulfide to sustain chemoautotrophy for hours without environmental input (Arndt et al., 1998; Girguis and Childress, 2006).

The unique nature of vestimentiferan symbioses has encouraged many physiological and biochemical studies of host and symbionts (for review, see Stewart and Cavanaugh, 2006). Until recently, however, little was known about the metabolic rates of these symbioses. Vent tubeworms usually die within several hours at one atmosphere, making it difficult to study whole-animal processes at atmospheric pressure. To address this problem, investigators developed a shipboard flow-through high-pressure respirometry system (HPRS) that maintains chemoautotrophic symbioses at in situ pressures, simultaneously delivers dissolved sulfide and oxygen at rates sufficient to support the high metabolic rates of the organisms, and concurrently measures changes in metabolite flux via a membrane inlet mass spectrometer (Girguis et al., 2000, 2002; Girguis and Childress, 2006; Fig. 1B). This technique allows for well-controlled respirometric experiments and is very effective at keeping deep-sea organisms alive both on board ship and in the laboratory. Using this system, they found that Riftia actively takes up nitrate—a first for any animal—that is reduced by the symbionts to provide ammonia for growth and biosynthesis (Girguis et al., 2000), and that it eliminates hydrogen ions—the primary waste product of symbiont metabolism—at unprecedented rates via numerous H^+-ATPases (Girguis et al., 2002).

View larger version (35K): [in this window] [in a new window]   Figure 1. (A) Photograph of a Ridgeia piscesae aggregation in situ. Photograph courtesy of V. Tunnicliffe, University of Victoria. (B) Schematic of the high-pressure respirometry system (HPRS). Sterilized (0.2-µm filter) seawater is pumped into the equilibration column, where it is mixed with gasses and compounds to mimic diffuse vent effluent. This solution is delivered to three high-pressure metering pumps, which generate 27.5 MPa pressures in the temperature-controlled pressure aquaria located between the pumps and the backpressure valves. Aquarium effluents are directed toward a stream-selection system that sends each stream to analysis and sampling.

Collectively, however, these studies focus largely on the metabolic responses of vestimentiferans to the vent environment. In some respects, all the prior physiological or biochemical studies of Riftia have been "black box" approaches, studying biochemical activities in vitro; whole-animal metabolic activity with little insight into the underlying molecular regulation; or gene and protein expression with little environmental context. Although the recent genomic, proteomic, and transcriptomics studies in this and other hydrothermal vent organisms have provided valuable insight into gene expression and biochemical potential, these studies used freshly collected specimens as their source material, many of which had spent several hours or more in conditions that were markedly different from their in situ environment (e.g., high oxygen concentrations and low pressures, Table 1). In short, such data cannot be allied to organismal activity at environmentally relevant conditions.

In light of the paucity of data on the molecular mechanisms underlying vestimentiferan symbiotic interactions, in particular during high metabolic activity, we conducted a series of experiments that employ the HPRS to ally metabolic rates with host gene expression during high net productivity in the vestimentiferan tubeworm Ridgeia piscesae Jones, 1985. Ridgeia piscesae is a tubeworm that flourishes at vents in the Pacific Northwest (Jones, 1985). It has widely variable morphologies that appear related to its local geochemical milieu. The "short and fat" form of Ridgeia (Fig. 1A), used in this study, is physiologically and morphologically very similar to Riftia (Jones, 1985; Flores et al., 2005; Carney et al., 2006; Urcuyo et al., 2003, 2007), but is found in more vigorous diffuse flows and can exhibit higher activity and growth (DeBovoise and Taghon, 1988). We chose to work with Ridgeia because of its amenability to shipboard manipulations (individuals are modest in size and very active), and its genetic and biochemical similarity to Riftia (the two species are phylogenetically allied, have similar hemoglobins, and likely host the same symbionts; Rouse, 2001; Flores et al., 2005; DiMeo et al., 2001). Also, in situ observations suggested that Ridgeia aggregations are capable of extremely rapid growth rates (J. Delaney, University of Washington, pers. obs.). The experiments and data presented here examine gene expression in the Ridgeia host trophosome (in particular the bacteriocytes) during periods of high primary productivity, in order to identify the mechanisms employed by these hosts to support high symbiont chemoautotrophic productivity.

	Materials and Methods

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Animal collection
Ridgeia piscesae tubeworms were collected during a research expedition from August to September 2006 to the Mothra (47°55.4'N by 129°06.4'W) and Main Endeavour vent fields (47°57.0'N by 129°5.82'W) along the Juan de Fuca ridge. Tubeworms were collected via the DSV Alvin from a depth of about 2300 m, brought to the surface in a thermally insulated container, and promptly moved to ice-cold seawater. We specifically collected the "short and fat" Ridgeia morphotype from active sulfide chimneys. Tubeworms that weighed between 5 and 20 g and were responsive to touch were immediately placed into flow-through, high-pressure respirometry aquaria for experimentation.

Experimental apparatus and design
During all respirometry experiments, tubeworms were maintained in the high-pressure respirometry system (HPRS; as described in Girguis and Childress, 2006). The aquaria were maintained at 27.5 MPa (4000 psi) by high-pressure pumps (American Lewa, Inc.) and backpressure valves. All experiments were maintained at 15 °C (a temperature measured near the base of Ridgeia aggregations in situ; Urcuyo et al., 2007). To simulate the seawater chemistry found in situ, 0.2-m filter-sterilized seawater was pumped into an acrylic gas equilibration column and bubbled with carbon dioxide, hydrogen sulfide, oxygen, and nitrogen to achieve the desired concentrations of dissolved gas. For the experiment outlined here, we maintained R. piscesae at conditions previously shown to support high primary productivity by Riftia (5.5 mmol l^–1 dissolved inorganic carbon; 215–230 µmol l^–1 H₂S; 150 µmol l^–1 O₂; 40 µmol l^–1 NO₃^–; pH 5.9; 15 °C; 27.5 MPa; Girguis and Childress, 2006). In the absence of data on Ridgeia physiology, we chose to replicate the chemical conditions that stimulated net carbon fixation in Riftia, which we believe is a reasonable representation of conditions found on the Juan de Fuca chimneys where these specimens were collected. At the end of the experiments, the worms were quickly removed from the pressure vessels; dissected into gill, vestimentum, body wall, and trophosome sections (taking care to selectively harvest the lobules containing the bacteriocytes); and flash-frozen in liquid nitrogen for subsequent RNA extraction (see below). In general, samples could be flash-frozen within 5 min of depressurization.

Determination of metabolite uptake rates
To determine substrate uptake rates, seawater was collected pre- and post-aquarium for chemical analyses. For total CO₂ (CO₂) and O₂ concentrations, 30 ml of effluent was added to N₂-purged 50-ml serum vials containing 3 ml of concentrated NaOH. CO₂ concentrations were measured by adjusting the pH to 3 using degassed HCl, and measuring PCO₂ in the headspace using a blood gas analyzer (Capni-Con II, Cameron Instruments). O₂ concentrations were measured using a gas chromatograph with a thermal conductivity detector (Agilent 6890N, as in Childress et al., 1984). For H₂S concentrations, 1.5 ml of effluent was preserved with 0.227 mol l^–1 zinc acetate and kept frozen and in the dark for later analysis. Subsequently, H₂S concentrations were determined using the method of Cline (1969). Cline’s "B" reagent consisted of 0.6% FeCl and 0.4% N,N-dimethyl-p-phenylenediamine in 50% HCl. A standard curve was generated using sodium sulfide concentrations varying between 1 µmol l^–1 and 1 mmol l^–1. All samples were measured on a Smart spectrophotometer (BioRad, Hercules, CA) at 670 nm. pH was determined on board ship using a double-junction pH electrode and high-precision pH meter (Radiometer, Inc.).

RNA extractions
Flash-frozen trophosome tissues were quickly cut into 1-cm pieces on a dry-ice-cooled stainless steel dissecting tray and placed immediately into pre-chilled (–20 °C) RNAice (Ambion Inc.) and allowed to equilibrate overnight at –20 °C. One gram each of both trophosome and plume from experimental and control specimens of R. piscesae was used to extract total RNA according to manufacturer’s guidelines (MaxiPrep total RNA extraction kit, Qiagen, Inc.). After extraction, total RNA was quantified on a microscale UV spectrophotometer (Nanodrop, Inc.) and quality checked by gel electrophoresis on an RNA microchip (Agilent, Inc.). Subsequently, Poly- (A)⁺ RNA was isolated using oligo-(dT)-chromatography, and eluted into Tris-EDTA buffer, pH 7 (Sigma, Inc.).

cDNA synthesis and EST library construction
A 500-µg sample of total RNA from pooled trophosome tissues was sent to the Joint Genome Institute (DOE, Walnut Creek, CA) for cDNA synthesis, library construction, and sequencing. Briefly, polyadenylated RNA was reverse transcribed with superscript reverse transcriptase using dT primer (5'-GACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTTVN-3'). cDNA was synthesized with E. coli DNA ligase, E. coli DNA polymerase I, and E. coli RNaseH. Ends were repaired with T4 DNA polymerase. An SalI adapter (5'TCGACCCACGCGTCCG-3' and 5'-P04-CGGACGCGTGGG-3’) was ligated to cDNA, and the product was digested with NotI. Digested cDNA was gel-purified, directionally ligated into SalI- and NotI-digested pCMVsport6, then transformed into ElectroMAX T1 DH10B cells. The transformation stock was plated onto 254 x 254-mm dry bioassay agar plates using glass beads. The plates were incubated for 18 h at 37 °C, then colonies were picked using a QPix2 XT colony robot picker and placed into 384-well microtiter dishes containing LB medium and 7.5% glycerol. Sequence template was prepared by rolling-circle amplification. Di-deoxy terminator sequencing was performed using BigDye terminator chemistry and an ABI 3100 DNA sequencer (Applied Biosystems, Foster City, CA) on 11,520 unique clones in both the 5' and 3' directions.

Sequence analysis
Sequence reads were called via ABI and PHRED version 0.020425.c sequence analysis software and were manually verified. Sequences were trimmed and edited in Sequencher ver. 4.1 (GeneCodes), and identified using the BLAST-based analysis program BLASTX (National Center for Bioinformatics Information; NCBI; Altschul et al., 1997), which compared our sequence to six non-redundant peptide sequence databases (GenBank CDS translations, RefSeq Proteins, PDB, SwissProt, PIR, and PRF). Sequences were then manually annotated on the basis of significant hits to annotated proteins (E value < 10^–10). BLAST was run between the fasta file containing all of the sequences that passed quality standards, and the KOG database (Tatusov et al., 2003). Sequences with KOG E values of < 10^–10 were categorized and quantified according to the functional category of the homologous gene.

	Results

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Metabolite flux
Specimens of Ridgeia piscesae were successfully maintained in our high-pressure respirometry system (HPRS) for 6 days under in situ environmental conditions (6 days was the duration of the expedition, though tubeworms have been maintained for up to 4 months in previous incubations). Worms exhibited little or no evidence of net carbon uptake during the first 6 h of incubation (similar to Riftia, Girguis et al., 2000). Subsequently H₂S, CO₂, O₂, and NO₃^– uptake increased, as well as proton equivalent elimination, indicating that the tubeworms were engaged in active chemoautotrophic sulfide oxidation and carbon fixation (Table 2). H₂S uptake rates averaged 5.47 ± 0.37 µmol·g^–1·h^–1, also comparable to those of Riftia (Girguis and Childress, 2006). CO₂, O₂, and NO₃^– uptake rates averaged 3.51 ± 1.76, 13.7 ± 1.9, and 1.42 ± 0.714 µmol·g^–1·h^–1 respectively. Proton equivalent elimination, previously shown to be a good indicator of chemoautotrophic function (Girguis et al., 2002), was also high, averaging 16.1 ± 2.6 µeq·g^–1·h^–1, with a peak of 47.5 µmol·g^–1·h^–1. The worms also deposited new tube near their gills at a rate of about 1 cm·day^–1 during these experiments (n = 11). These uptake rates persisted for greater than 66 h, and were maintained throughthe end of the experiment (at which point the worms were rapidly depressurized and flash-frozen). These metabolite uptake rates and tube growth rates indicate that R. piscesae thrived under our experimental conditions.

View larger version (31K): [in this window] [in a new window]   Figure 2. KOG analysis depicting all hits to the eukaryotic KOG database (Tatusov et al., 2003) with E values of greater than 10-10. KOG hits were categorized according to their homologs in the database. Ridgeia expressed sequence tags were highly diverse in function, but expression of protein biosynthesis genes, metabolic genes (in particular energy production and acid-base regulation) and signal transduction genes was very high.

	Discussion

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These metabolite flux rates also provide significant context for our gene expression libraries. Because of the ability to rapidly preserve tissues post-experimentation, the ESTs recovered in our libraries represent genes expressed by R. piscesae bacteriocytes (and other trophosomal tissues) during high chemoautotrophic activity (Fig. 2; Table 3). These ESTs provide a glimpse into host molecular processes underlying tremendous primary productivity. With respect to metabolism and homeostasis, we have recovered genes that encode for enzymes involved in acid-base regulation and inorganic carbon acquisition (in particular carbonic anhydrase, or CA). This finding corroborates previous studies—from biochemical assays to gene sequencing—that emphasize the importance of CA in enabling vestimentiferans to cope with the highly variable changes in environmental pH, as well as with the rapid production of protons resulting from symbiont sulfide oxidation (Kochevar and Childress, 1995; Goffredi et al., 1999a, b; De Cian et al., 2003; Sanchez et al., 2007). In particular, sulfate and proton equivalents are the primary end products of symbiont sulfide oxidation (Girguis et al., 2002), and active vestimentiferan symbionts generate protons at unprecedented rates (up to 119 µeq·g·h^–1; Girguis et al., 2002). Additionally, vestimentiferans must acquire inorganic carbon and transport it to the trophosome, while concurrently eliminating protons at rates sufficient to prevent metabolic acidosis. CA plays a significant role in transporting inorganic carbon (Goffredi et al., 1999a, b), while also buffering and indirectly transporting protons from the bacteriocytes and trophosome into the vascular blood, where gill H⁺ ATPases eventually eliminate them into the environment (Girguis et al., 2002). We also found high representation of genes involved with oxygen and sulfide binding, namely hemoglobin alpha and beta subunits, as well as a hemerythrin (an iron-containing non-heme protein that binds oxygen). Blood- and tissue-borne binding proteins have been shown to increase the transport of oxygen (and in this case sulfide; Arp and Childress, 1983), as well as the rate of diffusion into tissues (Sidell, 1998). All together, expression of these genes suggests that Ridgeia supports high productivity by further facilitating the diffusion of inorganic carbon, oxygen, and sulfide into the bacteriocytes, and the elimination of protons out of the bacteriocytes.

The majority of the genes recovered from the library, however, are implicated in mediating cell stress and governing cell regulation (Fig. 2, Table 3). Heat-shock proteins (such as the HSP70 that was recovered in our libraries) have been shown to be expressed during host-cell signaling cascades that occur during bacterial infection (Davies et al., 2006). We also identified a Bat1 homolog belonging to the DEAD-box family of RNA-binding proteins, previously demonstrated to be an important mediator of inflammation (Alcock et al., 2001). The identification of genes potentially encoding for viral precursors is very intriguing, as little is known about the role that viruses play in hydrothermal vent systems. A recent study showed that viruses are found in high densities at hydrothermal vents and likely cause significant microbial mortality (Ortmann and Suttle, 2005). However, the role of viruses in chemoautotrophic symbioses remains to be examined, though they could be a major factor in tubeworm mortality.

In addition to cell stress response and viral protein precursors, many genes were found that are known to be involved with regulating cell cycle, apoptosis, and cell growth; these genes include ubiquitin and homologs for skpA and RF-C activator 1, known mediators of cell cycle and replication in Drosophila (Murphy, 2003; Tsuchiya et al., 2007). Development and cell patterning genes such as a brachyenteron-like homolog, important in insect development and a member of the MADS-box family of transcription factors, implicated in development and differentiation in Rhizobium infections of root nodules, were also recovered (Heard and Dunn, 1995; Messengy and Dubois, 2003). Although the regulation of bacteriocyte development has not been described for any vestimentiferan, morphological studies suggest that a complicated bacteriocyte and symbiont cell cycle occurs in these associations (Bright and Sorgo, 2003). In Riftia, the endosymbionts appear to change from a rod-like morphotype near the axial blood vessel to larger, rotund cocci at the periphery of the trophosome lobule. The host bacteriocytes also change along with the symbionts, having the morphological appearance of undergoing apoptosis near the periphery of the lobules. It has been suggested that a significant fraction of the symbiont population may also be digested at any given time by the host (Bright and Sorgo, 2003).

These morphological observations suggest that the expressed gene repertoire in the trophosome may be responsible for far more than simply enhanced substrate delivery. Many of these described cell cycle and stress response pathways require well-established signaling cascades, and this study finds expression of such genes (such as a homolog to the neuropeptide growth hormone allatotropin as well as several involved with phosphorylation-based cellular signaling, see Table 3; Elekonich and Horodyski, 2003; Cooper et al., 2006). In such a closed and intimate association as that between hydrothermal vent tubeworms and their bacterial symbionts (e.g., bacterial symbionts in the adult host are never in contact with the external milieu), regulation of bacteriocyte and symbiont cell density and division is likely paramount to the association’s survival. The host must promote chemoautotrophic activity by its symbionts while preventing symbiont overgrowth to its detriment. Genes involved with regulating cell stress, cycle, and signaling are likely to be important in mediating these processes. Ongoing experiments are employing quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and mRNA capture to better quantify the changes in host bacteriocyte gene expression between individuals of Ridgeia exhibiting differences in net chemoautotrophy.

The data presented here focus on the suite of genes expressed by the host bacteriocytes and associated tissues during elevated chemoautotrophic activity by the intracellular symbionts. Although this illustrates how the host might sustain substrate availability while potentially regulating host transcriptome or symbiont cell cycle, it provides an incomplete story in the absence of data on symbiont gene expression. We are currently developing approaches to recover and sequence symbiont mRNA from these same experimental organisms, and to ally them to host gene expression. The genes recovered from genomes of two chemoautotrophic symbionts (Riftia pachyptila and Ruthia magnifica; Newton et al., 2007; Robidart et al., 2008) can help us identify their mode of action. Microbial-mediated apoptosis has been shown to be an important function in both symbiotic (Foster and McFall-Ngai, 1998; Heddi et al., 2005) and pathogenic associations (Hofman et al., 2004; Guiney, 2005; Zhang and Bliska, 2005) and may play an important role in these vestimentiferan symbioses as well. Other enhanced COG (clusters of orthologous groups) categories in the Riftia symbiont genome corroborate the indications of high metabolic rates and cell turnover in this study. As with the host bacteriocyte transcriptome, quantitative and higher resolution analyses will enable us to better understand the mode of communication between host and symbiont and the methods of regulating chemoautotrophic function and host and symbiont growth.

Although model systems like the insect-bacteriocyte, squid light organ, and Rhizobium/root nodule associations have provided insight into the types of genes and proteins that are involved in regulating host-symbiont cell cycle and cell-cell signaling (Gage, 2004; Heddi et al., 2005; Moran et al., 2005; Visick and Ruby, 2006), no model system has yet been identified to study metazoan-chemoautotrophic symbiont interactions. The nature of vestimentiferan associations—namely their efficacy as primary producers and their amenability to shipboard experimentation—make them an effective model system for studying these interactions. Despite the added challenges of working with complex experimental apparatus, the benefits of studying these associations in the carefully controlled conditions on board ship cannot be undervalued. Resulting from the first study of a chemoautotrophic symbiosis (or any deep-sea organism) that couples metabolite flux and transcriptomics, the approaches and results presented here have challenged our preconceptions about these associations, but have illustrated the potential range of experiments and wealth of information that can be gathered in such studies. The expressed genes recovered so far represent a fraction of a larger pool whose members are currently being sequenced, but already offer insight into the types of mechanisms that may be regulating these tubeworm symbioses. We believe that coupling experimental physiology with genomics and proteomics offers the best means by which we can further our understanding of these extraordinary organisms, and promises to be an exciting avenue of present and future research.

	Acknowledgments

 
This project was supported in part by the National Science Foundation (OCE-10293854 to P. Girguis). We thank the Joint Genome Institute for their phenomenal support and effort in constructing and sequencing EST libraries for Ridgeia piscesae and Riftia pachyptila. We are extremely grateful to the members of the Girguis lab, who were invaluable during these experiments. In particular, special thanks to Helen White for her assistance during the shipboard experiments. Thanks to Bryan Baclaski for proofreading assistance. We are also grateful to the crews of the R/V Atlantis and DSV Alvin during cruise AT15-9 during August-September, 2006.

	Footnotes

 
Received 5 December 2007; accepted 11 March 2008.

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