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Keywords:

  • 454-Pyrosequencing;
  • catalyzed reporter deposition-fluorescence in situ hybridization;
  • hydrothermal vents;
  • Ridgeia piscesae ;
  • symbiosis;
  • vestimentiferans

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

A large proportion of the faunal biomass in hydrothermal vent ecosystems relies on symbiotic relationships, with bacteria as a source of nutrition. Whereas multiple symbioses have been observed in diverse vent hosts, siboglinid tubeworms have been thought to harbour a single endosymbiont phylotype affiliated to the Gammaproteobacteria. In the case of the Northeast Pacific vestimentiferan Ridgeia piscesae, two previous studies suggested the presence of more than one symbiont. The possibility of multiple, and possibly habitat-specific, symbionts in R. piscesae provided a potential explanation for the tubeworm's broad ecological niche, compared with other hydrothermal vent siboglinids. This study further explored the diversity of trophosome bacteria in R. piscesae using two methodological approaches not yet applied to this symbiosis. We carried out 454-pyrosequencing on trophosome samples from 46 individual worms and used catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) to verify the presence of the major groups detected in the pyrotag data. Both methods yielded inconsistent and sometimes contradictory results between sampling sites, and neither provided irrefutable evidence for the presence of symbionts other than the expected Gammaproteobacteria. We therefore conclude that the other adaptive mechanisms must be considered to explain the broad physico-chemical niche occupied by the different growth forms of R. piscesae.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Hydrothermal vent environments host highly productive faunal communities relying on primary production by chemosynthetic microorganisms (Corliss et al. 1979; Jannasch & Wirsen 1979; Karl et al. 1980). Whereas free-living microbial communities represent an important source of organic carbon for suspension- and deposit-feeders, the bulk of the faunal biomass at most vent sites is supported by associations with symbiotic chemolithoautotrophic bacteria (Cavanaugh 1994; Watsuji et al. 2012; Ponsard et al. 2013). At Eastern Pacific vents, symbioses are dominated by large populations of siboglinid tubeworms. These gutless polychaetes host symbionts in an organ known as the trophosome (Cavanaugh et al. 1981; Felbeck 1981). Most studies have detected a single, specific endosymbiont that is common to this group of worms (Edwards & Nelson 1991; Feldman et al. 1997). In contrast, other symbiont-bearing invertebrates known from deep-sea reducing habitats (vents, cold seeps, and whale and wood falls), such as mytilid mussels (Distel 1995; Fiala-Medioni et al. 2002), alvinocarid shrimp (Zbinden et al. 2008; Petersen et al. 2010) and provannid snails (Suzuki et al. 2005; Urakawa et al. 2005), host phylogenetically and metabolically diverse chemosynthetic partners. Investigation of the phylogenetic position of siboglinid symbionts has revealed two clusters corresponding to either cold seep- or vent-hosted organisms (Di Meo et al. 2000), between which sequence divergence is around 4.3% (Vrijenhoek 2010).

There is some evidence for a more diverse trophosomal symbiotic assemblage in the Northeast Pacific siboglinid tubeworm Ridgeia piscesae. Early ultrastructural studies of R. piscesae trophosomes suggested that similar symbionts are found across worm morphotypes but that two morphologically distinct bacteria could occur within a single host (de Burgh et al. 1989). More recently, using terminal-restriction fragment length polymorphism (t-RFLP), Chao et al. (2007) detected the expected gammaproteobacterial phylotype plus two novel phylotypes from the same class, together with one Alphaproteobacteria and one Bacteroidetes. The goal of the present study was to pursue these investigations and explore the diversity of the bacteria within the R. piscesae trophosome using pyrosequencing and catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Pyrosequencing has not previously been used for screening endosymbiont diversity in vestimentiferans and is therefore considered exploratory.

Material and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Sample collection

Samples of Ridgeia piscesae were collected during three separate research expeditions in July 2010 onboard the R/V Atlantis, using the submersible Alvin, in July 2011 onboard the R/V Thomas G. Thompson, using the remotely operated vehicle ROPOS, and in June 2013 onboard the R/V Thomas G. Thompson using an Oceaneering Millennium Plus remotely operated vehicle. In 2010 and 2011, individuals of the two most extreme morphotypes of the tubeworm, known as the ‘short-fat’ and the ‘long-skinny’ morphotypes (Fig. 1), were sampled from two vent sites on Axial Volcano and four in the Main Endeavour vent field (Table 1). Smaller samples of the two morphotypes were collected from the Main Endeavour vent field in 2013. Samples were transported to the surface in sealed, separated bioboxes to prevent contamination between samples and from ambient seawater. Once on board, samples intended for pyrosequencing were pre-processed in a 5 °C cold room: the bodies of the worms were carefully removed from their tubes and cleaned with 70% ethanol, individually packed and frozen at −80 °C. For CARD-FISH, the tubes were removed and the bodies were cleaned as described previously. For the 2010 and 2011 individuals, the bodies were dissected and subsamples of tubeworm trophosome were fixed as described by Dubilier et al. (1995). For the 2013 samples, the bodies were fixed whole according to the previous protocol with some modification: the whole bodies were incubated in 4% paraformaldehyde/0.1 m phosphate-buffered saline (PBS) for 18 h at 4 °C. After three rinses in filtered water, they were gradually dehydrated and stored in 70% ethanol at 4 °C until sectioning. Some specimens were fixed without rinsing to assess potential epibiotic contamination.

Table 1. Description and location of sampling sites.
sampling site IDtubeworm morphotypevent sitelatitudelongitudedepth (m)max. temp (°C) at plumecollection dateanalysis technique(s)no. of individuals analysed
LF10AV1bLong-skinnyAxial Volcano (Hollywood Flats 1)45°56.147′ N129°58.888′ W1518na10 JulyPyrosequencing5
HF10AV2bShort-fatAxial Volcano (Hollywood Flats 2)45°56.155′ N129°58.893′ W15174.110 JulyPyrosequencing & CARD-FISH5 & 3c
LF10AV2bLong-skinnyAxial Volcano (Hollywood Flats 2)45°56.156′ N129°58.890′ W15172.010 JulyPyrosequencing & CARD-FISH5 & 3c
LF10TPbLong-skinnyMain Endeavour (TP)47°56.971′ N129°5.854′ W21972.4July-10Pyrosequencing5
HF10HUbShort-fatMain Endeavour (Hulk)47°57.007′ N129°5.824′ W219014.010 JulyPyrosequencing & CARD-FISH5 & 3c
LF10HUbLong-skinnyMain Endeavour (Hulk)47°57.007′ N129°5.825′ W21912.510 JulyPyrosequencing & CARD-FISH5 & 3c
HF11GRbShort-fatMain Endeavour (Grotto)47°56.953′ N129°5.903′ W218821.611 JulyPyrosequencing5
HF11LBbShort-fatMain Endeavour (Lobo)47°56.965′ N129°5.900′ W219112.211 JulyPyrosequencing5
image

Figure 1. Examples of typical sampling sites. (A) Aggregation of the ‘short-fat’ morphotype of Ridgeia piscesae. (B) Zoom out showing a black smoker in the surrounding area. (C) Habitat of the ’long-skinny’ morphotype of R. piscesae. Here, no shimmering is visible.

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454-Pyrosequence library construction

DNA was extracted from approximately 25 mg of tissue using the DNeasy Blood and Tissue Kit (Qiagen, Carlsbad, CA, USA), following the manufacturer's instructions, from a total of 40 tubeworm trophosomes (Table 1). DNA was purified and concentrated using the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer's instructions, and 20 μl of DNA with a concentration of 20 ng·μl−1 or higher was sent to the Plateforme d'Analyses Génomiques (Institute of Integrative and Systems Biology, Laval University, Quebec City, QC, Canada). The hypervariable region V1-V3 of the bacterial SSU rRNA gene was amplified by PCR using Takara Ex Taq premix (Fisher Scientific, Toronto, ON, Canada). PCR reactions were performed in a final volume of 50 μl containing 25 μl of Premix Taq, 1 μm of each primer and sterile Milli-Q H2O to up to 50 μl (a list of the primers is available in Supporting information, List S1). PCR conditions were as follows: after a denaturing step of 30 s at 98 °C, samples were processed through 30 cycles consisting of 10 s at 98 °C, 30 s at 55 °C and 30 s at 72 °C. A final extension step was performed at 72 °C for 4 min 30 s. Following amplification, samples were purified using magnetic AMPure Beads (Beckman Coulter Genomics, Mississauga, ON, Canada) to recover PCR amplicons, separating them from contaminants. Samples were adjusted to 100 μl with EB buffer (Qiagen), to which 63 μl of beads were added. Samples were mixed and incubated for 5 min at RT. Using a magnetic particle concentrator (MPC), the beads were pelleted against the wall of the tube and supernatant was removed. The beads were washed twice with 500 μl of 70% ethanol and incubated for 30 s each time. Supernatant were removed and beads were allowed to air dry for 5 min. Tubes were removed from the MPC and 15 μl of EB buffer was added. Samples were vortexed to resuspend the beads. Finally, using the MPC, the beads were pelleted against the wall once more and supernatant were transferred to a new clean tube. DNA concentrations in sample were quantified by Nanodrop and mixed in equal amounts. Pyrosequencing was performed using a 454 GS-FLX DNA Sequencer with the Titanium Chemistry (Roche, Branford, CT, USA) according to the procedure described by the manufacturer.

Pyrosequencing read analysis

All data processing and analyses were performed using the software program MOTHUR. Raw pyrosequences were checked for different quality criteria. Reads with an average quality score below 27 (Kunin et al. 2010), containing an error in the forward primer sequence at the beginning of the read (Sogin et al. 2006), containing one or more ambiguous bases (Ns) (Sogin et al. 2006; Huse et al. 2007), or shorter than 250 base pairs (De Leon et al. 2012) were eliminated. A set of unique reads was created and aligned against the SILVA-based bacterial reference alignment (Pruesse 2007) provided by MOTHUR using the Needleman–Wunsch pairwise alignment algorithm (Needleman & Wunsch 1970). A pre-clustering step was applied to group sequences differing by <2% (corresponding to five mismatches for a 250-base-pair sequence) (Huse et al. 2010). Potential chimeras were identified using the program UCHIME (Edgar et al. 2011) and removed from the dataset. Because there was a large range between the minimum and maximum number of reads found in our samples, the three samples with the lowest numbers of reads (one sample from HF10AV2 with 807 reads, one sample from HF10HUb with 2294 reads, and one sample from LF10AV1b with 1583 reads) were eliminated from further analyses. Singletons (unique sequences) were also eliminated. Then, the number of reads across samples was standardized by subsampling, based on the lowest number of sequences (3593) found in any of the remaining 37 samples. The remaining sequences were classified using the Silva template database with 1000 bootstrap iterations. The command ‘phylotype’ was used to generate a file listing the sequences affiliated to each taxon at the phylum, class, order, family and genus levels. A shared file, which described the number of times each taxon was observed in all samples, was generated.

Nucleotide sequence accession numbers

The pyrosequence reads have been deposited in the NCBI Short Read Archive under accession number SRA058565.

Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH)

Fixed tissues of individual worms from the 2010 and 2013 collections were embedded in paraffin and sectioned (5 μm thickness) onto glass slides. The pre-hybridization treatments were performed as previously described (Dubilier et al. 1995) with the following modifications: sections were deparaffinized in CitriSolv (Fisher Scientific), a less toxic alternative to xylene, and the post-fixation step in 3.7% formaldehyde was omitted. CARD-FISH was carried out as described by Blazejak et al. (2005) with the following horseradish peroxidase (HPR)-labelled oligonucleotide probes: EPSY549, specific to the Epsilonproteobacteria, GAM42a, covering most Gammaproteobacteria, EUB338, targeting the domain Bacteria as a positive control, and NON338, a complementary negative control. Sequences for these general probes were obtained through the probeBase website (Loy et al. 2007). For each probe, formamide concentration was optimized using a range of different concentrations to get the best signal with the highest formamide concentration (most stringent conditions possible) (Table 2). The fluorescently labelled tyramides were prepared as described by Pernthaler et al. (2004) with the Alexa Fluor 488, 555 (Molecular Probes - Invitrogen, Burlington, ON, Canada). A few sections were hybridized without a probe to control for background fluorescence. For multiple hybridizations, the CARD-FISH protocol was repeated with the same sections with different probes and dyes as described in Blazejak et al. (2005). Slides were imaged under epifluorescence and confocal illumination, using a Leica Leitz DMRB fluorescent microscope or a Nikon C1 Plus confocal microscope.

Table 2. Oligonucleotide probe description.
probespecificitysequence (5′–3′)positiona[formamide] (%, v/v)creference
  1. aPosition in the 16S rRNA gene of Escherichia coli unless indicated otherwise.

  2. bPosition in the 23SrRNA gene of E. coli.

  3. cIn hybridization buffer. Numbers in bold indicate the concentration used in this study.

  4. dNon-labelled probe.

EUB338BacteriaGCTGCCTCCCGTAGGAGT338–35550-55-60Amann et al. (1990)
EPSY549EpsilonproteobacteriaCAGTGATTCCGAGTAACG549–56650-55-60Lin et al. (2006)
GAM42aGammaproteobacteriaGCCTTCCCACATCGTTT1027–1043b50-55-60Manz et al. (1992)
NON338Negative controlACTCCTACGGGAGGCAGC338–35525-30-35-40-55Widdel & Bak (1992), Wallner et al. (1993)

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

454-pyrosequence library

For the 40 tubeworm trophosomes sampled in 2010 and 2011, a total of 645,009 reads were obtained through pyrosequencing. After quality filtering and removing the three less abundant samples, 513,860 high-quality pyrosequences remained, representing 3022 unique sequences. A total of 1825 singletons were eliminated from further analysis and standardization left 132,941 sequences, of which 893 were unique. Eleven different phyla were detected but only two represented more than 1% of the sequence library: the Proteobacteria and Bacteroidetes. Within the Proteobacteria, which represented 97.0% of the sequences, Gammaproteobacteria were clearly the most abundant class, followed by Epsilonproteobacteria, Deltaproteobacteria, Alphaproteobacteria, and Betaproteobacteria (Fig. 2). The other phyla detected, accounting for 0.1% of the sequence library, included Actinobacteria, Firmicutes, Chloroflexi, Spirochaetes, Acidobacteria, Cyanobacteria, Verrucomicrobia, and the candidate divisions BD1-5 and TM7.

image

Figure 2. Relative abundance of the phyla accounting for >1.0% of the pyrosequence library constructed from the trophosomes of 37 individuals of Ridgeia piscesae. As the Proteobacteria accounted for 97.0%, this phylum was divided into the five classes detected.

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Results were not consistent between locations. For the six sites sampled in 2010, sequences belong to classes other than the Gammaproteobacteria constituted 10–30% of the sequence libraries (Fig. 2), whereas for the two sites sampled in 2011 (HF11GRb & HF11GBb in Fig. 2) and the 2013 sites (data not shown), non-Gammaproteobacteria made negligible contributions to the sequence libraries.

Bacteria detection in R. piscesae trophosome

Multiple hybridizations GAM42a confirmed the dominant presence of members of with Gammaproteobacteria within the trophosome of single R. piscesae in all individuals analyzed. Dual hybridization with EPSY549 and GAM42a, suggested a minority presence of dispersed Epsilonproteobacteria in the trophosome tissue in the 2010 samples (Fig. 3A), and an almost negligible presence in the 2013 samples (data not shown). To assess the binding specificity of EPSY549 the probe was hybridized in parallel with the general bacteria probe (EUB338) using tissue sections from 2010 samples (Fig. 4A,B). The high concentration of Gammaproteobacteria (Fig. 3A) made it difficult to assess whether Epsilonproteobacteria were also detected with EUB338 (Fig. 4A). Although the majority of the Epsilonproteobacteria appeared to co-localize with EUB338, there was some indication of non-specific binding (Fig. 4A,B, arrows). Hydridization with the NON338 probe yielded some very bright points, mostly co-localizing with nuclei of epithelial cells (Fig. 4C,D), whereas the remainder of the EPSY549 signal was localized in the central and median zone of the trophosome lobes (Figs 3A and 4A). The non-specific signal was most likely the result of binding of the probe to other cellular components rather than mispairing with non-target sequences (Wallner et al., 1993). Hybridization with another negative control (ECO1459 targeting the non-hydrothermal vent species Escherichia coli) resulted in a similar, non-specific signal (data not shown).

image

Figure 3. Double-probe catalysis reporter deposition fluorescent in situ hybridization of 5-μm sections of Ridgeia piscesae dissected trophosomes, with (A) EPSY549 (red), (B) merged GAM42a (green) and DAPI (blue) signals. Scale bars: 50 μm. Image taken under a Leica Leitz DMRB fluorescent microscope.

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image

Figure 4. Double-probe catalysis reporter deposition fluorescent in situ hybridization of the same region of the dissected trophosome of an individual Ridgeia piscesae. (A) EPSY549, (B) merged EUB338 and DAPI signals, (C) NON338, (D) DAPI. Arrows represent some of the non-specific signals in (A, B) and (C,D). Scale bars: 200 μm. Image taken under a Nikon C1 Plus confocal microscope.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

Symbiotic associations between bacteria and eukaryotes are widespread in the biosphere and dominate faunal biomass in reducing habitats such as hydrothermal vents, cold seeps, and whale and wood falls, where the host bridges oxic and anoxic zones, facilitating access to oxidants and reductants for the chemoautotrophic symbionts (Cavanaugh et al. 2006; Watsuji et al. 2012; Ponsard et al. 2013). Whereas the association of vent organisms from many different families with multiple bacterial phylotypes is well documented (Distel 1995; Fiala-Medioni et al. 2002; Suzuki et al. 2005; Urakawa et al. 2005; Petersen et al. 2010), vent tubeworms have been thought to host only one symbiont phylotype shared across the entire group (Edwards & Nelson 1991; Feldman et al. 1997; Markert et al. 2007; Robidart et al. 2008). In the case of Ridgeia piscesae, two previous studies suggested the presence of more than one bacterial phylotype in the trophosome (de Burgh et al. 1989; Chao et al. 2007). The de Burgh et al. (1989) study was based entirely on morphological examination of bacterial cells within trophosome tissue, without molecular evidence that the different morphologies corresponded to phylogenetically distinct bacteria. Chao et al. (2007) provided molecular evidence, but the authors recognized that their conclusion, based solely on T-RFLP data, could have been influenced by contamination from bacteria associated with the worm tubes or the environment in which they were collected (Chao et al. 2007). In this study, we used 454-pyrosequencing and CARD-FISH to further explore the possibility of multiple endosymbionts in R. piscesae. The pyrosequencing approach has rarely been used to investigate endosymbiont diversity, and as the rare biosphere detected by this technique is more likely to contain artefacts (Huse et al. 2010; Kunin et al. 2010; Tedersoo et al. 2010), we applied very strict quality filtering to reduce background contamination as well as a high number of replicates to improve data comparability (Zhou et al. 2011). Pyrosequencing results from a previous study of free-living bacterial associated with R. piscesae assemblages (Forget & Juniper 2013) provide some background for interpretation of data presented here, particularly with respect to possible contamination. Nevertheless, the results obtained from pyrosequencing should be considered exploratory and the detection of rarer or unexpected bacterial lineages, i.e. other than Gammaproteobacteria, need be confirmed by PCR-independent methods such as CARD-FISH. This point is reinforced by the inconsistent pyrosequencing results obtained here for tubeworms collected from similar physico-chemical habitats. The latter observation rules out any simple explanation based on the worm's physiological requirements. The presence of non-symbiont sequences resulting from pyrosequencing artefacts or environmental contamination (external bacteria) represents a more parsimonious explanation for these inconsistencies.

The dominant presence of Gammaproteobacteria in R. piscesae trophosome tissue from both habitats was confirmed by CARD-FISH. This result is not surprising, as the previously known siboglinid endosymbionts are members of the Gammaproteobacteria. The affiliation of the most abundant genera to the genus Methylomicrobium is doubtful. We used MOTHUR and the Silva alignment to compare the classification of a SSU rRNA gammaproteobacterial sequence identified as R. piscesae endosymbiont [accession number U77480 (Feldman et al. 1997)]. The near-full length of the gene was identified as a member of the family Sedimenticola, which corresponds to the affiliation of the R. pachyptila endosymbiont, Candidatus Endoriftia persefone (based on the EzTaxon-e server (Kim et al. 2012) but the V1-V3 region of the sequence was classified as the genus Methylomicrobium from the family Methylococcaceae. Okubo et al. (2012) assessed phylogenetic drifts of pyrosequence read classification and suggested that assignment at the genus level is affected by read length. Such classification errors can lead to incorrect conclusions about the ecological role of the community investigated.

Members of the Epsilonproteobacteria have been found within the epibiotic community of the galatheid crab Shinkaia crosnieri (Watsuji et al. 2012), the alvinocaridid shrimp Rimicaris exoculata (Zbinden et al. 2008; Petersen et al. 2010; Guri et al. 2012), the alvinellid polychaetes Paralvinella sulfincola, Paralvinella palmiformis and Alvinella pompejana (Haddad et al. 1995; Campbell et al. 2001; Alain et al. 2002; Pagé et al. 2004), as well as the siboglinid polychaetes R. pachyptila and R. piscesae (López-García et al. 2002; Kalanetra & Nelson 2010). Epsilonproteobacterial endosymbionts have been previously detected in provannid gastropods from the genus Alviniconcha (Urakawa et al. 2005), and in pectinodontid gastropods from the genus Pectinodonta (Zbinden et al. 2010), but pyrosequencing results from our 2010 samples (30 individual worms) represent the first indication of their presence in vent siboglinids. The most abundant genus detected in our pyrosequence library, Sulfurovum, was also the most abundant group detected in the free-living bacterial communities associated with R. piscesae (Forget & Juniper 2013). This result could indicate environmental contamination of the trophosome. The low relative abundance of epsilonproteobacterial pyrotags in the 2010 data and their absence in sequence data from 2011 and 2013 samples rule out a systematic presence and role in the tubeworm's nutrition, and reinforce the external contamination explanation. Epsilonproteobacteria have been shown to contribute to other invertebrate-prokaryote symbioses at hydrothermal vents. In the case of R. exoculata, the closest relative to their epsilonproteobacterial symbionts was also a member of the genus Sulfurovum (Petersen et al. 2010). A trophic role was suggested for epsilonproteobacterial epibionts in the case of S. crosnieri and R. exoculata (Watsuji et al. 2012; Ponsard et al. 2013).

CARD-FISH revealed the expected dominance of Gammaproteobacteria in the trophosome tissue of all specimens plus a low-level presence of cells hybridizing to the Epsilonproteobacteria probe in the 2010 samples. The latter were diffusely distributed throughout the trophosome tissue sections, and the NON-probe indicated some to be non-specific. Given the contradictory results obtained with the Epsilonproteobacteria probe in samples from the 2 years (and locations) and the fact that, when detected, putative Epsilonproteobacteria were very scarce and similar in abundance to points of non-specific hybridization, we could not definitely confirm the presence of Epsilonproteobacteria in the trophosome.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

This study is the first to explore potential symbiotic diversity within a siboglinid tubeworm through 454-pyrosequencing. The surprisingly high diversity of taxonomic groups revealed by pyrosequencing must be handled carefully: the majority of the genera detected had very low frequency. Molecular methods based on rRNA genes can be sensitive to the relative abundance of organisms and biased toward those with higher gene copy numbers (Farrelly et al. 1995; Crosby & Criddle 2003; Hoshino et al. 2008). The preponderance of Gammaproteobacteria within tubeworm trophosomes, and of one particular phylotype, could have limited the detection of less abundant groups/phylotypes in other studies. The next most abundant class of bacteria for which there was some pyrotag evidence was the Epsilonproteobacteria. However, 454-pyrosequencing results were inconsistent among the locations (and years) sampled in this study, with respect to the presence of Epsilonproteobacteria, and CARD-FISH results were even less supportive. We therefore conclude that there is currently no irrefutable evidence to support previous suggestions of that R. piscesae hosts multiple symbionts. It therefore appears to be necessary to consider other mechanisms that could permit R. piscesae and its symbionts to occupy the broad range of physico-chemical conditions that are exploited by the different growth forms of this tubeworm. For example, metagenomic analysis and related profiles of gene expression may provide insight into how R. piscesae symbionts and their host interact with their external environment.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information

The authors would like to thank Dr Bob Chow for access to his confocal microscope and for his expertise and valuable time training of N.L.F. and M.P. We are also grateful to the team of Wax-it Histology Services for their collaboration during the preparation of the tissue sections and to Candice St. Germain for sharing her samples and for insightful discussions. We thank the crews of the R/V Atlantis and R/V Thomas G. Thompson as well as the pilots of the submersibles Alvin and ROPOS. This research was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to S.K.J. and a Canadian Healthy Oceans Network (NSERC Canada) grant to Dr Verena Tunnicliffe. During this study, N.L.F. benefitted from an NSERC graduate scholarship, a Montalbano Scholars Fellowship, a Dr Arne Lane Graduate Fellowship, a Commander Peter Chance MASC Graduate Fellowship, an Alfred and Adriana Potvin Graduate Scholarship in Ocean Sciences, a W. Gordon Fields Memorial Fellowship, a Charles S. Humphrey Graduate Student Award, and a Maureen De Burgh Memorial Scholarship.

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  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
maec12169-sup-0001-ListS1.pdfPDF document38KList S1. List of primers used for pyrosequencing.

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