Use of the SSH method for the study of symbiosis
We used SSH on different tissues from a single individual to look for genes involved in the functioning of the symbiosis because it is not possible to obtain aposymbiotic adult Riftia pachyptila. The body wall was used as a reference tissue to find specific proteins expressed in the gills (main metabolite exchange organ with the milieu) and in the trophosome (organ that houses the symbiotic bacteria). We then focused our attention on some chosen transcripts for a quantitative analysis. The remaining unidentified sequences (many of which could correspond to 3'UTR portions of cDNA) could prove interesting. Their future identification will require either a RACE approach or hybridization on a full length cDNA library.
Efficiency of the SSH method
All the 10 sequences that were more closely studied by quantitative PCR showed differential expression in agreement with the subtractive libraries where they were found. A transcript obtained in a given library showed the highest expression in the expected tissue, as evidenced by checking the result with rapid PCR validation (Fig. 2) and from several individuals by quantitative PCR (Fig. 3). In addition, although constitutively expressed in all cells of all tissues, only one tubulin transcript sequence was obtained from the BR-BW library, and one actin sequence from the BW-TR library. This demonstrates the adequate subtraction of these common sequences. However, some sequences are sometimes highly represented, possibly indicating a subtraction that was not as efficient. ccox I, for example, could not be eliminated in the BR-BW cDNA pool, but this may be due to the fact that it was more expressed in the branchial plume than in the body wall tissue. Strangely however, we recovered the RpCAbr transcript from the BW-BR library (Table 3) although it was 109-fold less abundant in the body wall than in the branchial plume tissues (Fig. 3).
As noticed by Ji and coworkers , SSH PCR favors highly differentially expressed genes. From our quantitative PCR results, some transcripts showed such high differential expression (e.g. RpCAbr in the branchial plume compared to the other tissues, and RpCAtr in the trophosome compared to the other tissues). These authors suggest that the primary application of SSH PCR should be the detection of dramatic alteration of gene expression, as it is for example the case for gene expression profiling of two different tissues. Our use of SSH for comparing pairs of tissues seems very appropriate.
Proteins degradation and turnover in the branchial plume tissue
Some transcripts were preferentially expressed in the branchial plume tissue. Relative quantification of the cathepsin transcript (a degradation enzyme found in lysosomes) revealed a more important expression in the branchial plume tissue, compared to the body wall (protected by the tube) and the trophosome. The plume is the only organ in direct contact with sea-water, and thereby strongly exposed to hydrogen sulfide and other toxic molecules such as heavy metals which are abundant in the hydrothermal vent environment. Electron-dense organelles (EDO) seem to be very common in tissues of sulfide-adapted marine annelids. Such structures have previously been observed in both the Riftia pachyptila epidermal body wall  and the branchial plume organ (Ann Andersen, personal communication). Arp and coworkers  hypothesized that EDO structures could actually be secondary lysosomes. This could be in agreement with our results of cathepsin expression, found to be highest in this highly exposed tissue. Julian and coworkers  showed that even in sulfide-tolerant organisms like the annelid Glycera dibranchiata, sulfide exposure poisons the mitochondria, leading to depolarization that is not reversible. Lysosomes could degrade mitochondria that have been damaged by sulfide exposure. Besides, we also found a Rab5 GDP/GTP exchange factor (BRbwC22), which is a member of the Ras superfamily of GTPases. This protein, involved in vesicle trafficking, is known to be located in early endosomes that precede the formation of lysosomes.
Interestingly, another pathway of degradation could also be involved since we obtained a Valosin-Containing Protein (VCP, BRbwC21), which is required in ubiquitin-proteasome degradation . The existence of two VCP transcripts has recently been demonstrated in an annelid, the earthworm Eisenia fetida . Our sequence shows the best homology scores with the predicted VCP protein sequence of Strongylocentrotus purpuratus [Expect = 0.014] and the eVCP-1 isoform from Eisenia fetida [Expect = 0.025] which is ubiquitously expressed in this worm .
If EDOs do represent autophagic degradation of organelles as suggested by Arp and coworkers , rapid replacement of organelles should take place . The high expression level of ribosomal 16S, an essential gene for the translation of mitochondrial messenger RNAs into proteins, and the presence of some transcripts linked to transcription (BRbwC19 and 20, Table 2) is then consistent with a high protein turnover in this tissue.
Sulfide oxidation with the concomitant production of ATP by the mitochondria of the annelid Arenicola marina has been shown . However, no protein sequence was found here that would suggest a similar property of R. pachyptila mitochondria.
Hydroxylamine reductase protein
Formerly known as prismane, the hydroxylamine reductase is a member of the Hybrid-Cluster Protein (HCP) family and is thought to play a role in nitrogen metabolism. It catalyses the reduction of hydroxylamine to form ammonia using NADH. In rat liver mitochondria, this enzyme is firmly attached to the mitochondrial membrane  and its activity can prevent hydroxylamine to inhibit mitochondrial respiration . However, blasts of our sequence did not match with the few eukaryotic sequences available but resulted in 100 bacteria sequences hits. The best ones are those of the Actinobacteria Salinispora arenicola [Expect = 5e-19] and the α-proteobacteria Rhodospirillum rubrum [Expect = 5e-19]. Because this sequence did not match any sequence in the Riftia symbiont genomic database, it could be contamination from bacteria living close to the branchial plume of the worm.
Major Vault Protein gene expression
The Major Vault Protein (MVP) (100 kDa) is the major protein component of vaults, ribonucleic particles of 13MDa. Some studies established that vaults could be involved in nucleocytoplasmic transport of ribosomes and/or mRNA . This could be coherent with our results of 16S expression obtained on the branchial plume tissue in which probable high transcription levels of this protein occur. Other studies indicate the participation of MVP in drug resistance mechanisms where it could act as a nucleocytoplasmic and vesicular transporter of drugs and/or metabolites to transport them to exocytotic vesicles or proton pumps [34, 35]. It could be evidenced that MVP gene in Mytilus edulis was predominantly expressed in epithelia-rich tissues such as the gills and digestive gland and could be involved in multixenobiotic resistance . In our study, MVP transcript is preferentially expressed in the branchial plume tissue compared to the body wall, while no MVP transcript was detected in the trophosome samples. The presence of such a protein in the branchial plume tissue may be used to temporarily immobilize toxic molecules before they are processed.
Chitinase gene expression
Interestingly, a chitinase precursor was recovered as a branchial plume specific transcript. A previous report indicated chitinase activity in the opisthosome and branchial plume of R. pachyptila . Chitin is a major component of the tube of R. pachyptila, produced by specialized glands located in the body wall and the vestimentum . Chitinase activity was suggested to be involved in tube growth and tube shape modifications . A chitinase sequence was recently discovered in the hydroid cnidarian Hydractinia  and a possible role of chitinase enzyme in pattern formation and allorecognition was suggested. Interestingly, the transcript was exclusively expressed in ectodermal tissues of the animal, and the authors also suggested a possible role in host defense against pathogens. Such a hypothesis could be interesting to explore given our quantitative PCR experiments because we only could amplify this transcript from cDNA from the branchial plume, the only organ in contact with the environmental sea water.
Tissue-specific expression of different carbonic anhydrases
Our quantification analyses showed a higher abundance of the RpCAbr transcript in the branchial plume compared to the trophosome (present at very low levels) and the body wall tissues. In contrast, the RpCAtr transcript was very abundant in the trophosome compared to the branchial plume (medium levels) and the body wall tissues. Fluorescent In Situ Hybridization confirmed the co-expression 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 CA cDNAs with vertebrate and non-vertebrate CA protein sequences revealed the conservation of most amino acids involved in the catalytic site, indicating that the two proteins are probably functional if the cDNAs are translated .
Myohemerythrin, T-cell receptor, and unidentified transcripts from the trophosome library
A complete coding sequence obtained from the TR-BW library (contig 17, Table 4) showed a very high homology score with a myohemerythrin sequence from the Sipuncula Sipunculus nudus [GenBank:CAG14944] (Expect = 1e-11). The complete Riftia sequence has an open-reading frame of 120 amino acids. Myohemerythrin is an oxygen-binding protein that participates in the storage of oxygen in muscles. Such a protein could be involved in the regulation of cadmium levels in the gut of the annelid Nereis diversicolor . In Hirudo medicinalis, it would have indirect antibacterial properties by regulating free iron availability to deprive bacteria of iron essential for their growth .
Both TCR and TRbwC27 cDNAs showed specific expression in the trophosome tissue where they could be essential. The TCR transcript first caught our attention because it matched a sequence fragment coding for a T-cell receptor, which is a complex of integral membrane proteins that participates in the activation of T-cells in response to the presentation of an antigen. The trophosome is mostly composed of bacteriocytes which house bacterial cells in intracellular vacuoles and cellular recognition may be very important for the functioning of this tissue. As for the TRbwC27 it is a large fragment of 273 nucleotides which is highly represented in our subtracted library but did not reveal reliable Blastx homology E-values.