Siboglinidae Tubes as an Additional Niche for Microbial Communities in the Gulf of Cádiz—A Microscopical Appraisal

Siboglinids were sampled from four mud volcanoes in the Gulf of Cádiz (El Cid MV, Bonjardim MV, Al Gacel MV, and Anastasya MV). These invertebrates are characteristic to cold seeps and are known to host chemosynthetic endosymbionts in a dedicated trophosome organ. However, little is known about their tube as a potential niche for other microorganisms. Analyses by scanning and transmission electron microscopy showed dense biofilms on the tube in Al Gacel MV and Anastasya MV specimens by prokaryotic cells. Methanotrophic bacteria were the most abundant forming these biofilms as further supported by 16S rRNA sequence analysis. Furthermore, elemental analyses with electron microscopy and energy-dispersive X-ray spectroscopy point to the mineralization and silicification of the tube, most likely induced by the microbial metabolisms. Bacterial and archaeal 16S rRNA sequence libraries revealed abundant microorganisms related to these siboglinid specimens and certain variations in microbial communities among samples. Thus, the tube remarkably increases the microbial biomass related to the worms and provides an additional microbial niche in deep-sea ecosystems.


Introduction
Chemosynthetic fauna is widely distributed and often found in deep-sea areas of active fluid seepage where oxygen levels are normally low, such as in hydrothermal vents and cold seeps. Yet, they can also be found in other reduced environments, such as whale and wood falls [1][2][3][4][5]. While the composition of the seepage fluids is variable, some (micro)organisms have adapted to use some of the most abundant constituents as their energy and/or carbon source, i.e., methane and sulfur compounds. These chemosynthetic organisms sustain these ecosystems by acting as primary producers and supplying the higher trophic levels with nutrients [6,7]. They also provide the hard substrate that

Imaging of endosymbionts
During transmission and scanning electron microscopy, worm tissues were only observed in the Al Gacel MV (see supplementary data Figure S1) and Anastasya MV samples ( Figure 2). The other  Table 1.

Scanning Electron Microscopy (SEM) and Energy-Dispersive X-Ray Spectroscopy (EDX) Analysis
While specimens from El Cid MV and Anastasya MV were dehydrated without prior fixation, specimens from Al Gacel MV were fixed in 2.5 % (w/v) glutaraldehyde to maintain the native organic structures. After washing several times with phosphate-buffered saline, a dehydration series was performed (15%, 30%, 50%, 70, 80%, 90%, 95%, and 100% aqueous ethanol solution), followed by hexamethyldisilazane (HMDS; Sigma-Aldrich, Germany) in order to avoid drying artifacts. Samples were mounted on SEM sample holders and sputtered with Au-Pd (13.9 nm for 120 s). They were further visualized in a SEM LEO 1530 Gemini (Zeiss, Oberkochen, Germany) combined with an INCA X-ACT EDX.

Fluorescent Staining of Chitin Tubes
Tubes of specimens recovered from the Al Gacel MV were stained with calcofluor white (Merck, Darmstadt, Germany) to identify the chitin tube. Following previous staining of the samples, they were fixed on a slide and embedded in paraffin followed by a graded ethanol series (100%, 90%, 70%, and 50%). Afterwards, one drop of staining and one drop of KOH 10% were placed onto the slide with the sample. The samples were examined under normal light and a UV filter with an excitation ranges between 450 and 490 nm of a Zeiss Axioplan microscope (Zeiss, Oberkochen, Germany).

DNA Extraction and Amplification of Bacterial and Archaeal 16S rRNA Genes
Between 10 and 15 specimens (bulk of empty tubes and worms inside of tubes) from El Cid MV, Bonjardim MV, Al Gacel MV and Anastasya MV were used for this analysis. Total DNA was isolated with Power Soil DNA Extraction Kit (MO BIO Laboratories, Carlsbad, CA, USA) according to manufacturer's instructions. Bacterial amplicons of the V3 -V4 region were generated with the primer set S-D-Bact-0341-b-S-17 / S-D-Bact-0785-a-A-21, with added Illumina adapter overhang nucleotide sequences (forward primer: 5 -TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG  GGN GGC WGC AG-3 ; reverse primer: (5 -GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA  GGA CTA CHV GGG TAT CTA ATC C-3 ) [31]. Likewise, archaeal amplicons of the V3 -V4 region were generated with the forward primer based on Arch514Fa (5 -TCG TCG GCA GCG TCA GAT GTG  TAT AAG AGA CAG GGT GBC AGC CGC CGC GGT AA-3 ) and the reverse primer (5 -GTC TCG  TGG GCT CGG AGA TGT GTA TAA GAG ACA GCC CGC CAA TTY CTT TAA G-3 ) [32]. The PCR reaction mixture for bacterial DNA amplification, with a total volume of 50 µl, contained 1 U Phusion high fidelity DNA polymerase (Biozym Scientific, Oldendorf, Germany), 5% DMSO, 0.2 mM of each primer, 200 µM dNTP, 0.15 µL of 25 mM MgCl 2 , and 25 ng of isolated DNA. Furthermore, PCR protocol for bacterial DNA amplification was: initial denaturation for 1 min at 98 • C, 25 cycles of 45 s at 98 • C, 45 s at 60 • C, and 30 s at 72 • C, and a final extension at 72 • C for 5 min.
The PCR reaction mixture for archaeal DNA amplification was similarly prepared but contained 1 µL of 25 mM MgCl 2 and 50 ng of isolated DNA. PCR protocol for archaeal DNA amplification was: initial denaturation for 1 min at 98 • C, 10 cycles of 45 s at 98 • C, 45 s at 63 • C, and 30 s at 72 • C, 15 cycles of 45 s at 98 • C, 45 s at 53 • C, and 30 s at 72 • C, and a final extension at 72 • C for 5 min. PCR products were purified using the GeneRead Size Selection Kit (QIAGEN GmbH, Hilden, Germany).

Bioinformatic Processing of Amplicons
300 Paired-end (300PE) sequencing of the amplicons was performed in the Göttingen Genomics Laboratory (Göttingen, Germany). Paired-end sequences were merged, and sequences containing unresolved bases and reads shorter than 305 base pairs (bp) were removed using PANDAseq v2.11 [33], employing the PEAR algorithm v0.9.8 [34]. Non-clipped forward and reverse primer sequences were removed by employing cutadapt v1.15 [35]. QIIME 1.9.1 was used to process the amplicon sequences [36]. The sequences were dereplicated and checked for chimeric sequences (de novo). Sequences were clustered at 97% sequence identity to operational taxonomic units (OTUs). The taxonomic classification of the OTU sequences was performed against the SILVA database 132 employing the assignment method implemented in Mothur [37]. Extrinsic domain OTUs, chloroplasts, and unclassified OTUs were removed from the dataset. Sample comparisons were performed at the same surveying effort, utilizing the lowest number of sequences by random subsampling (30,563 reads for bacteria, 4080 reads for archaea). Beta-diversity was calculated using Unifrac statistics [38] to determine the distances between samples. Principal coordinates analysis (PCoA) plots representing the data was visualized using EMPeror tool [39]. R programming was used to construct heatmaps representing the relative abundances of bacterial and archaeal communities in each sample. The paired-end reads of the 16S rRNA gene sequencing were deposited in the National Center for Biotechnology Information (NCBI) in the Sequence Read Archive SRR8944123 with the accession number PRJNA533037.

Samples and in Situ Variables' Measurement
Siboglinid specimens were recovered from different mud volcanoes at sites where reduced sediment was observed. Exact location of the samples, as well as data collected from the ROVs' sensors (CH 4 and CTD) and are shown in Table 1. El Cid MV and Bonjardim MV specimens were sampled from grey mounds ( Figure 1B,C). The El Cid MV siboglinid-sample was collected from the first 5 cm of a sediment push-core, while the Bonjardim MV sample, which was found in a mud breccia with a strong hydrogen sulfide smell, was collected using a suction sampler. Siboglinid specimens recovered from the Al Gacel MV were located in a pockmark, beneath an AOM-derived carbonate and facing an active bubbling seepage ( Figure 1D) [40]. Furthermore, Anastasya MV specimens were obtained from a field of Beggiatoa-like biofilms ( Figure 1E). All specimens were about 100 µm width and not more than 15 cm in length. Their tubes had a light-brownish color. No intensive morphological identification could be made; however, based on their size and external appearance they are likely Siboglinum sp. or Sclerolinum sp. specimens.

Imaging of endosymbionts
During transmission and scanning electron microscopy, worm tissues were only observed in the Al Gacel MV (see supplementary data Figure S1) and Anastasya MV samples ( Figure 2). The other samples consisted of empty tubes. SEM micrographs from one specimen of Anastasya MV revealed the posterior region of the worm ( Figure 2A) with a segmented opisthosoma and the trophosome ( Figure 2B). A hole in the trophosome exposed abundant bacteria inside ( Figure 2C,D). These bacteria were cocci of ca. 0.5 µm in diameter ( Figure 2D).

Structure and Composition of the Tubes
The fluorescent stain calcofluor white is an indicator for polysaccharides such as chitin, which is part of the organic matrix of siboglinids' tubes. Sections of empty tubes from the Al Gacel MV expressed high fluorescence when observed under UV-light with a 09 Zeiss filter, with excitation wavelength ranges between 450 and 490 nm ( Figure 3). Furthermore, an external microbial layer deattached from the tube (most likely due to handling of the sample) was slightly fluorescent ( Figure  3B).

Structure and Composition of the Tubes
The fluorescent stain calcofluor white is an indicator for polysaccharides such as chitin, which is part of the organic matrix of siboglinids' tubes. Sections of empty tubes from the Al Gacel MV expressed high fluorescence when observed under UV-light with a 09 Zeiss filter, with excitation wavelength ranges between 450 and 490 nm ( Figure 3). Furthermore, an external microbial layer de-attached from the tube (most likely due to handling of the sample) was slightly fluorescent ( Figure 3B).
SEM micrographs revealed transversal-segmented tubes, which were covered by minerals (from El Cid MV, Figure 4A), a thick biofilm (from Al Gacel MV, Figure 4B) or putative remains of microbial extracellular polymeric substances or EPS (from Anastasya MV, Figure 4C). Disrupted tubes revealed their composition of multiple concentric layers between 6 and 10 µm in thickness ( Figure 4D). Some of the layers displayed a filamentous matrix, with attached globular particles of ca. 200 nm in diameter ( Figure 4E). Layers consisting of these particles show a significant silica signal in EDX analysis (see supplementary data Figure S2). Other layers contained significant amounts of iron, sulfur and calcium, without notable differentiation between them. Detailed interpretation of EDX-analysis is discussed in supplementary data. Furthermore, microbial cells were observed in the internal surface of the tube from Al Gacel MV ( Figures 4D and 5G,H). A model of the different layers observed in a tube is shown in Figure 4F. SEM micrographs revealed transversal-segmented tubes, which were covered by minerals (from El Cid MV, Figure 4A), a thick biofilm (from Al Gacel MV, Figure 4B) or putative remains of microbial extracellular polymeric substances or EPS (from Anastasya MV, Figure 4C). Disrupted tubes revealed their composition of multiple concentric layers between 6 and 10 µ m in thickness ( Figure 4D). Some of the layers displayed a filamentous matrix, with attached globular particles of ca. 200 nm in diameter ( Figure 4E). Layers consisting of these particles show a significant silica signal in EDX analysis (see supplementary data Figure S2). Other layers contained significant amounts of iron, sulfur and calcium, without notable differentiation between them. Detailed interpretation of EDXanalysis is discussed in supplementary data. Furthermore, microbial cells were observed in the internal surface of the tube from Al Gacel MV ( Figures 4D and 5G,H). A model of the different layers observed in a tube is shown in Figure 4F.

Microbial Biofilm of the Tubes
TEM and SEM micrographs from the Al Gacel MV revealed a high microbial colonization of the outside surface of the tube (Figures 4B and 5). The biofilm was up to 10 and 20 µ m thick ( Figure 5A). Bacteria with intracytoplasmic membranes arranged as known for methanotrophic proteobacteria were the most abundant along the tube, forming densely packed cell-aggregations ( Figure 5A-C). Other microbial morphotypes were observed, i.e., prosthecate, rod shaped, helically shaped, and filamentous microorganisms ( Figure 5D-F). Rod-shaped microorganisms were also observed

Microbial Biofilm of the Tubes
TEM and SEM micrographs from the Al Gacel MV revealed a high microbial colonization of the outside surface of the tube ( Figures 4B and 5). The biofilm was up to 10 and 20 µm thick ( Figure 5A). Bacteria with intracytoplasmic membranes arranged as known for methanotrophic proteobacteria were the most abundant along the tube, forming densely packed cell-aggregations ( Figure 5A-C). Other microbial morphotypes were observed, i.e., prosthecate, rod shaped, helically shaped, and filamentous microorganisms ( Figure 5D-F). Rod-shaped microorganisms were also observed attached to the inside surface of the tube ( Figure 5G,H) Furthermore, some microorganisms appeared to be actively penetrating the chitin tube ( Figure 5I). Similarly, siboglinids' tubes from Anastasya MV under the TEM revealed a biofilm on the external tube face. However, the biofilm appeared to be in a degradation process, because cells appeared as "ghosts" (only cell walls, no cytosolic contents were visible; Figure 6). Remains of EPS forming similar cell-aggregations to the ones observed in Al Gacel MV tube indicate abundance of methanotrophic bacteria ( Figure 6A). Embedded remains of microorganisms inside the tube were also observed ( Figure 6B).

Prokaryotic Community Composition
Bacterial and archaeal 16S rRNA gene libraries revealed relative abundances of taxa typically found thriving in the water column, such as Acidobacteria, Actinobacteria, Bateriodetes, Chloroflexi, Thermoplasmata, Woesearchaeota, and Candidatus Nitrosopumilus (Figure 7) [41,42]. Sulfide-oxidizing bacteria are detected in all samples, with a high representation in the El Cid MV sample, being mostly Thiohalophilus and bacteria from the Thiotrichaceae family ( Figure 7A). Sedimenticola endosymbionts, which are also sulfide-oxidizing bacteria, are abundant in Al Gacel MV specimens, as well as Desulfobacterales sulfate-reducers. In fact, sulfate reducers are highly abundant (>15%) in Al Gacel MV and Anastasya MV samples, while in El Cid MV and Bonjardim MV they represent 3% of the total relative abundance ( Figure 7A). In Anastasya MV sample, Marine Methylotrophic group 2 (MMG-2) methanotrophic bacteria, and Desulfobacter sulfate-reducing bacteria are highly abundant ( Figure 7A). Additionally, Methylotenera methylotrophic bacteria taxa are also representative in Al Gacel MV (7%). In Al Gacel MV and Anastasya MV up to 50 % of the bacteria are represented by methane-oxidizing, sulfide-oxidizing and sulfate-reducing bacteria ( Figure 7C). Likewise, Chitinivibrionia (known chitin degraders) were detected in all our samples, especially in Anastasya MV ( Figure 7A).
The archaeal community profile was dominated by Woeserarchaeota (or DHVEG-6, Nanoarchaeota), followed by methane-oxidizing archaea (ANME-1 and ANME-2) as the second most abundant taxa, except in Anastasya MV where methanogens are slightly more abundant ( Figure  7B,D). Additionally, methanogenic archaea were homogeneous among the samples, except in the Al Gacel MV where they were almost absent ( Figure 7B).
Beta-diversity among the samples showed substantial differences in the microbial communities ( Figure 7C-D). The first and the second principal coordinates (PCoA1 and PCoA 2, respectively) revealed short distances between El Cid MV and Bonjardim MV bacterial communities ( Figure 7C), and between El Cid MV and Anastasya MV archaeal communities ( Figure 7D). Sulfide-oxidizing bacteria are detected in all samples, with a high representation in the El Cid MV sample, being mostly Thiohalophilus and bacteria from the Thiotrichaceae family ( Figure 7A).
Sedimenticola endosymbionts, which are also sulfide-oxidizing bacteria, are abundant in Al Gacel MV specimens, as well as Desulfobacterales sulfate-reducers. In fact, sulfate reducers are highly abundant (>15%) in Al Gacel MV and Anastasya MV samples, while in El Cid MV and Bonjardim MV they represent 3% of the total relative abundance ( Figure 7A). In Anastasya MV sample, Marine Methylotrophic group 2 (MMG-2) methanotrophic bacteria, and Desulfobacter sulfate-reducing bacteria are highly abundant ( Figure 7A). Additionally, Methylotenera methylotrophic bacteria taxa are also representative in Al Gacel MV (7%). In Al Gacel MV and Anastasya MV up to 50 % of the bacteria are represented by methane-oxidizing, sulfide-oxidizing and sulfate-reducing bacteria ( Figure 7C). Likewise, Chitinivibrionia (known chitin degraders) were detected in all our samples, especially in Anastasya MV ( Figure 7A).
The archaeal community profile was dominated by Woeserarchaeota (or DHVEG-6, Nanoarchaeota), followed by methane-oxidizing archaea (ANME-1 and ANME-2) as the second most abundant taxa, except in Anastasya MV where methanogens are slightly more abundant ( Figure 7B,D). Additionally, methanogenic archaea were homogeneous among the samples, except in the Al Gacel MV where they were almost absent ( Figure 7B).
Beta-diversity among the samples showed substantial differences in the microbial communities ( Figure 7C,D). The first and the second principal coordinates (PCoA1 and PCoA 2, respectively) revealed short distances between El Cid MV and Bonjardim MV bacterial communities ( Figure 7C), and between El Cid MV and Anastasya MV archaeal communities ( Figure 7D).

Endosymbionts in Siboglinidae Worms
Since siboglinids were discovered in the 1900s and described by Caullery in 1914 [43], researchers have collected data on their life history characteristics and, in particular, adaptations allowing them to survive in reduced environments at high hydrogen sulfide concentrations and low oxygen [12]. To date, it has been established that these tube-dwelling annelids harbor chemolithoautotrophic endosymbionts in the super-vasculated trophosome [19,20]. Those endosymbionts are facultative free-living bacteria which are acquired from the environment by the worms during their juvenile stage, when their guts are reduced [44,45]. Once they become adults, they have established a permanent mutualistic microbe-animal symbiosis, with the host lacking gut and acquiring organic carbon solely from their endosymbionts. This mechanism of obtaining endosymbionts horizontally from the environment has been also described in other animals [46].
Siboglinidae worms mostly harbor thiotrophic bacteria in their trophosomes [16], and only some punctual specimens have been reported to harbor methanotrophic endosymbionts instead, i.e., Siboglinum poseidoni recovered from central Skagerrak [22], and Sclerolinum contortum sampled at the Haakon Mosby MV [24] and the Gulf of Cádiz [23]. In the current study, endosymbionts from specimens collected in El Cid MV, Al Gacel MV and Anastasya MV were identified. Bacterial 16S rRNA genes from El Cid MV sample presented an OTU with 99 % similarity to a thiotrophic endosymbiont of Siboglinum worms recovered from Gemini MV in the Gulf of Cádiz (OTU_0) [47]. Likewise, Al Gacel MV worms revealed high abundance of an OTU with 98% similarity to Sedimenticola sp., a thiotrophic endosymbiont of Sclerolinum contortum (OTU_4) [48]. Furthermore, we observed bacteria inside of the trophosome ( Figure 2C,D) of a small siboglinid from the Anastasya MV (attempted to be classified as Siboglinum sp., due to its lack of girdles between the trophosome and opisthosoma; Figure 2A) [20]. Previous studies have also reported Siboglinum sp. worms in this volcano [49]. Bacterial 16S rRNA genes revealed that the most abundant methane-oxidizing bacteria in Anastasya MV specimens were related to Marine Methylotrophic Group 2 (MMG-2, Methylococcales; Figure 7A). MMG-2 bacteria have not previously been described as endosymbionts, but MMG-1 and MMG-3 [50]. The high presence of those sequences could indicate that these methanotrophs are actually acting as endosymbionts, as previous studies in Captain Arutyunov MV (Gulf of Cádiz) have reported the presence of these worms living in symbiosis with methanotrophic bacteria [23]. However, those sequences could also be related to the microbial remains observed onto the Anastasya MV tubes ( Figure 6) and therefore further studies discriminating between the tube and the tissue of siboglinids worms will be necessary.

The Tube as a New Microbial Niche
External and internal microbial colonization of siboglinid tubes has previously been described [25][26][27]. Light microscopy, SEM and TEM micrographs showed highly colonized tubes in Al Gacel MV specimens (Figures 3, 4B and 5). While microorganisms observed in the internal face of the tube seemed to be a thin microbial layer with isolated microorganisms (Figure 5H), externally, a thick microbial biofilm was composed of mostly methanotrophic bacteria forming cell aggregations ( Figure 5A-C), but also filamentous ( Figure 5D,E), prosthecate-and spirillum-shaped ( Figure 5F), and rod-shaped microorganisms were observed ( Figure 5G,H). Microbial 16S rRNA genes from the Al Gacel MV sample revealed the high abundance of bacteria related to Methylococcales (mostly MMG-2), possibly forming the characteristic microbial biofilm. Few sequence-reads were related to Hyphomonodaceae and prosthecate bacteria, which could explain the morphotypes observed on the external biofilm of Al Gacel MV worm ( Figure 5F). Rod-shaped bacteria could not be related to any microbial taxa since it is a highly common morphotype ( Figure 7A).
Al Gacel MV specimens represent an example of how the tubes of siboglinids provide a viable niche for microorganisms. In fact, microbial biofilms are known to be ecosystems themselves, capable of self-regulation in which all microorganisms are linked and provide each other with stable sources of nutrients and protection [51]. Those microorganisms increment the impact of siboglinid worms in the ecosystem, since they constitute part of the worms' microbiota and previous studies have pointed out the difference in the microbial composition of siboglinid tubes and the surrounding environment [27]. Consequently, the tube of siboglinids should be considered as an important niche, which increases the microbial biomass and provides a large source of microorganisms, which are part of the microbiota of the worms and ultimately the worm's holobiont [52].

Tube-Microbe Interaction
The tubes of all siboglinids all have in common that they produce a chitin matrix (Figure 3) secreted by the worm that is incorporated in the tube [18]. Since they are in contact with water and reduced sediments, tubes are rich in minerals and other inorganic compounds, which may vary depending on the environment [53]. High amounts of iron, calcium and sulfur compounds were detected in all the tubes with EDX analysis (supplementary Figure S2), indicating the precipitation of minerals such as pyrite (or ferrous sulfide), aragonite [17,[54][55][56], or even iron-silicates [57]. The presence of pyrite and aragonite in reduced environments is usually due to the anaerobic oxidation of methane (AOM) [8]. Microorganisms involved in AOM (sulfate-reducing bacteria and ANME archaea), as well as sulfur oxidizers (which oxidize pyrite), have been found at all sites ( Figure 7). Biomineralization of the tube due to the different microbial metabolisms leads to the embedding of such microorganisms inside of the tube (Figure 6B), by coating the external face of the tubes with precipitated minerals. Furthermore, microbial mineralization is accompanied by the precipitation of silica, which interacts with the iron forming iron-silicates and fills up the spaces between the chitin matrix ( Figure 4E), ultimately replacing the organic matter [17]. Consequently, composition of siboglinid tubes is principally characterized by microbial layers, chitin-silica layers, precipitated-minerals layers-replacing previous microbial biofilms-and external microbial biofilms. Thus, a model has been proposed showing the composition of the different layers given in the tube of a siboglinid worm based on our results and other studies ( Figure 4F) [17,[54][55][56].
Additionally, Chitinovibrionia chitin-degraders were detected mainly in Anastasya MV ( Figure 7A) and Al Gacel MV samples ( Figure 5I). This active participation of the biofilm on the tube's decay could indicate a switch in the microbial community, from a chemosynthetic-based microbial community-which coats and protects the tube-to a heterotrophic-based microbial community. Decay of the chemosynthetic-based biofilm was observed in the Anastasya MV tubes (Figures 4C and 6). Since active emission or hydrocarbon-rich fluids were detected at both sites (see Section 4.4), the decay of these certain tubes is not well understood and could be related to the life-cycle of the worm and not only dependent on the availability of reduced compounds. Additionally, handling of the sample during fixation could have led to the removal of microbial cells and only the EPS extracellular matrix has remained. Further studies focused on the life cycle of these biofilms, as well as their interaction with the worms and impact in the environment, are warranted.

Insights into Siboglinids' Microbiota
Siboglinidae worms do not only harbor microorganisms in their trophosome, but also on their tubes. Besides, rod-shaped bacteria were observed on an Anastasya MV worm, as potential epibionts (supplementary Figure S3). All these microorganisms associated with Siboglinidae specimens conform the microbiota (or microflora) of these invertebrates. This microbiota is part of its host, and the metabolisms driven by these microorganisms contribute to the total ecological impact of the worm on the environment. Worm and microbiota constitute therefore a unique ecological unit, sometimes referred to as holobiont [52]. Thus, in the same way the community of a mud volcano switches between chemosynthetic and non-chemosynthetic organisms depending on changes of the source of nutrients (i.e., seeped fluids versus organics from photic zone), we observed disparity in the total microbiota of siboglinids sampled from different mud volcanoes and sites with different seepage activity (Figure 7).
El Cid MV and Bonjardim MV specimens were recovered from sites where non-active emission of fluids was detected, and methane concentration was relatively low (70-90 nM and 50-65 nM, respectively; Sánchez-Guillamón et al., 2015) ( Figure 8) [58]. The site of El Cid MV from where worms were sampled, was surrounded by non-chemosynthetic fauna (shrimps, fish; Figure 8), while Bonjardim MV sampling was performed in an area where patches of reduced sediment (biofilm-like) and dead bivalves were observed (Figure 8). The sampled sediment with siboglinids from Bonjardim MV emanated a strong smell of hydrogen sulfide, potentially indicating the occurrence of anaerobic oxidation of methane (AOM) in the past. Likewise, the high relative abundance of sulfide oxidizers in El Cid MV samples may also indicate past AOM events ( Figure 7C). In fact, DNA related to ANME in both inactive sites was detected (El Cid MV and Bonjardim MV; Figure 7B). Furthermore, sulfate-reducing bacteria are much less abundant in these samples when compared to known active sites (i.e., Al Gacel MV and Anastasya MV; Figure 7A).
On the other hand, the Al Gacel MV and Anastasya MV sampling sites showed bubbling of gas methane hydrates (Figure 8) with methane concentrations as high as 191 nM at the time of Sclerolinum worm sampling [58]. At both sites a thick biofilm covering the tube of mainly methanotrophic bacteria was detected-in Anastasya MV specimens only remains of the biofilm were observed-and environmental 16S rRNA genes revealed a higher presence of methane-oxidizing and sulfate-reducing microorganisms in these samples ( Figure 7A,B). Since siboglinids normally colonize the oxic-anoxic interface in sites of fluids emission, they optimize at the same time the access to the seeped fluids for both aerobic methane-oxidizing bacteria and anaerobic sulfate-reducing bacteria, allowing them to co-exist in the same niche (the worm).
Interestingly, non-active (El Cid MV, Bonjardim MV) and active (Al Gacel MV, Anastasya MV) sites differ in the composition of their microbial communities ( Figure 7C,D). Therefore, our preliminary analyses indicate that seepage activity at these sites directly influence the composition of the microbial community ( Figure 7). Since DNA analysis in this study have been used to support imaging approaches and have preliminary results of siboglinids' total microbiota-without differentiating between tube's microbiota and worm-tissue's microbiota-further studies in that direction need to be performed including variation in the microbiota depending on the presence or absence of the living worms. and dead bivalves were observed (Figure 8). The sampled sediment with siboglinids from Bonjardim MV emanated a strong smell of hydrogen sulfide, potentially indicating the occurrence of anaerobic oxidation of methane (AOM) in the past. Likewise, the high relative abundance of sulfide oxidizers in El Cid MV samples may also indicate past AOM events ( Figure 7C). In fact, DNA related to ANME in both inactive sites was detected (El Cid MV and Bonjardim MV; Figure 7B). Furthermore, sulfatereducing bacteria are much less abundant in these samples when compared to known active sites (i.e., Al Gacel MV and Anastasya MV; Figure 7A).

Conclusions
Small Siboglinidae specimens appeared to have a higher microbial biomass related to them than previously realized. In addition to the chemosynthetic endosymbionts harbored inside their trophosome, specimens from Al Gacel MV and Anastasya MV revealed that the tube was colonized by a thick microbial biofilm. This external biofilm of the tubes was mostly composed of cell-aggregations of methanotrophic bacteria, but other morphotypes such as filamentous, prosthecate, spirillum-like and rod-shaped bacteria were also observed. Yet, this chemosynthetic-based biofilm seems to contribute to the biomineralization and silification of the tube, conditioning the different concentric layer given in siboglinid tubes. The microbial community on the tube can also participate in the degradation of the chitin tube by chitin-degrading bacteria. Additionally, a preliminary comparison of environmental 16S rRNA gene libraries showed different microbial communities among samples.