Science of the Total Environment 796 (2021) 149046

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Science of the Total Environment

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Characterization of tissue-associated bacterial community of two Bathymodiolus species from the adjacent cold seep and hydrothermal vent environments

Genmei Lin a, Jianguo Lu a,b,⁎, Zhilei Sun c,d, Jingui Xie a, Junrou Huang a, Ming Su a,b, Nengyou Wu c,d,⁎⁎ a School of Marine Sciences, Sun Yat-sen University, Zhuhai 519082, China b Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Zhuhai 519080, China c Key Laboratory of Gas Hydrate, Ministry of Natural Resources, Institute of Marine Geology, China Geological Survey, Qingdao 266071, China d Laboratory for Mineral Resources, Qingdao Pilot National Laboratory for Marine Sciences and Technology, Qingdao 266071, China

HIGHLIGHTS GRAPHICAL ABSTRACT

• Various bacterial community was asso- ciated with different tissues in seep and vent mussels. • A similar symbiotic gill-associated bac- terial population was found in the two deep-sea habitats. • Bacterial community in other tissues were different in two habitats without species variation. • Tissue-associated bacterial community may play multiple roles in element cy- cling. • The major putative function of gill- associated bacterial community was methane oxidation.

article info abstract

Article history: Deep-sea mussels are widely distributed in marine chemosynthetic ecosystems. Bathymodiolus platifrons and Received 5 May 2021 B. japonicus, occurring at both cold seeps and hydrothermal vents, have been reported to house exclusively Received in revised form 8 July 2021 methanotrophic symbionts in the gill. However, the comparison of microbiota associated with different tissues be- Accepted 10 July 2021 tween these two species from two contrasting habitats is still limited. In this study, using B. platifrons and B. japonicus Available online 15 July 2021 collected from the adjacent cold seep and hydrothermal vent environments, we sampled different tissues (gill, ad- Editor: Julian Blasco ductor muscle, mantle, foot, and visceral mass including the gut) to decipher the microbial community structure at thetissuescalebyemploying16SrRNAgene sequencing strategy. In the gill of both seep mussels and vent mussels, the symbiont gammaproteobacterial Methylomonaceae was the predominant lineage, and methane oxidation was Keywords: identified as one of the most abundant putative function. In comparison, abundant families in other tissues were Bathymodiolus mussel Pseudomonadaceae and Enterobacteriaceae in seep mussels and vent mussels, respectively, which may get involved Tissue-associated bacterial community in element cycling. The results revealed high similarity of community structure between two mussel species from Cold seep the same habitat. The gill showed distinctive bacterial community structure compared with other tissues within Hydrothermal vent thesameenvironment,whilethegillcommunitiesfromtwo environments were more similar. Remarkably struc- tural variations of adductor muscle, mantle, foot, and visceral mass were observed between two environments. This study can extend the understanding on the characteristics of tissue-associated microbiota of deep-sea mussels from the adjacent cold seep and hydrothermal vent environments. © 2021 Published by Elsevier B.V.

⁎ Correspondence to: J. Lu, School of Marine Sciences, Sun Yat-sen University, Zhuhai 519082, China. ⁎⁎ Correspondence to: N.Wu, Key Laboratory of Gas Hydrate, Ministry of Natural Resources, Institute of Marine Geology, China Geological Survey, Qingdao 266071, China. E-mail addresses: [email protected] (J. Lu), [email protected] (N. Wu).

https://doi.org/10.1016/j.scitotenv.2021.149046 0048-9697/© 2021 Published by Elsevier B.V. G. Lin, J. Lu, Z. Sun et al. Science of the Total Environment 796 (2021) 149046

1. Introduction been discovered in the northern and central OT recently (Sun et al., 2015, 2019; Xu et al., 2018; Cao et al., 2020; Li et al., 2021). In addition, Deep-sea bathymodiolin mussels are one of the most numerically the distance between the cold seeps and the active hydrothermal vents dominant macrofaunae fueled by symbioses with chemosynthetic mi- is only tens of kilometers (Zhang et al., 2019), thus provides us a pre- croorganisms in a wide range of ecosystems including sunken woods, cious opportunity to compare and link the microbial community associ- whale falls, cold seeps, and hydrothermal vents (Baco and Smith, ated with deep-sea mussels from these two extreme ecosystems. 2003; Pailleretetal.,2007; Levin et al., 2016), which is ecologically im- In this study, we characterized tissue-associated microbial portant as they could provide habitats for other animals and support community structure of two Bathymodiolus species (B. platifrons and highly productive animal communities through the dense mussel bed B. japonicus) collected from the geographically adjacent cold seep and formation (Sibuet and Olu, 1998; Bergquist et al., 2005; Xu et al., 2019). hydrothermal vent in the Okinawa Trough, in an attempt to address Genus Bathymodiolus (Bivalvia: Mytilidae), occurring worldwide at the following questions: (1) what are the relationships of associated cold seeps and hydrothermal vents, host chemosynthetic bacteria in microbial community between mussels from two environments? the gill where the flow of hydrothermal fluids or hydrocarbon seepage (2) What are the similarities and differences of associated microbial can provide a continuous supply of reduced substrates (e.g., methane, community between two mussel species within the same environ- hydrogen sulfide, and hydrogen), oxidants, and CO2 (Childress et al., ment? (3) What is microbial community structure associated with dif- 1986; Sogin et al., 2020). The gill endosymbiotic types mainly include ferent tissues? This study can provide a comprehensive description of methanotrophic, thiotrophic, or both (Fisher et al., 1993; Dubilier the microbiota (focusing on bacterial microbiome) features at the tissue et al., 2008). Thiotrophic bacteria are capable of using reduced sulfur scale, with the aim of shedding lights on the understanding on the char- compounds (such as sulfide and thiosulfate) as energy sources and acteristics of tissue-associated microbiota of two mussel species from carbon dioxide as the major carbon source (Distel et al., 1995; Arndt the adjacent cold seep and hydrothermal vent environments. et al., 2001). Hydrogen is also an energy source for thiotrophic symbi- onts of hydrothermal vent mussels (Petersen et al., 2011; Ikuta et al., 2. Material and methods 2016). Methanotrophic bacteria can utilize methane as their primary car- bon and metabolic energy source (Spiridonova et al., 2006; Szafranski 2.1. Sampling collection and tissue preparation et al., 2015). The symbiont types exhibit species specificity (Duperron et al., 2009). For example, vent mussel species B. azoricus and Deep-sea mussels studied in this work were collected from a B. puteoserpentis from the Mid-Atlantic Ridge (Fiala-Médioni et al., 2002; newly discovered cold seeping site named Station S11 on the west Kádár et al., 2005; Halary et al., 2008; Wendeberg et al., 2012), B. aff. boo- slope of the Okinawa Trough and the known Minami-Ensei Knoll merang from cold seep areas in the deep Gulf of Guinea (Duperron et al., hydrothermal field during the integrated environmental and geolog- 2011), as well as B. brooksi from seep habitats in the Gulf of Mexico (Raggi ical expedition of R/V Zhangjian carried out during September to et al., 2013; Picazo et al., 2019) all harbor a dual symbiosis with the October 2018 (Fig. 1). The Station S11 is an active mud volcano intracellular coexistence of both methane- and sulfide-oxidizing bacteria. with a diameter of about 160 m, the summit of which is about 35 m In comparison, B. childressi from the Gulf of Mexico contains only higher than the peripheral seafloor. The fluid seepage is still ongoing methanotrophic symbiont (Dattagupta et al., 2004; Duperron et al., and obvious bubble plume can be visible when sampling. The whole 2007), whereas B. thermophilus from hydrothermal vents of the East active area is estimated to be more than 1000 m2. Cold seep mussels PacificRise,B. septemdierum from hydrothermal vents of Japanese waters, were collected by the remotely operated vehicle (ROV) FCV3000 and B. aduloides from a cold seep in the South China Sea live in a symbiosis holding a self-made sampler at a depth of 896 m (DIVE ROV01-5). with sulfur-oxidizers only (Fisher et al., 1987; Fujiwara et al., 2000; Feng Hydrothermal vent mussels were collected from the Minami-Ensei et al., 2015; Ponnudurai et al., 2017b). Knoll hydrothermal field about 50 km away from Station S11 during B. platifrons and B. japonicus house exclusively methanotrophic sym- thesamecruise,withasamplingdepthof705m(DIVEROV02-1). bionts (Fujiwara et al., 2000; Barry et al., 2002). The habitats of most The mussels were gathered from about 200 m away from the central deep-sea mussel species are restricted to either seeps or vents, while vent and were often fixed to the massive hydrothermal barite or sul- B. platifrons and B. japonicus are distributed in both cold seep and hydro- fide basement (SI Fig. S1). thermal vent environments in West Pacific Ocean such as Sagami Bay, Once the mussels were brought to the surface, they were immedi- the Okinawa Trough, and the South China Sea (Miyazaki et al., 2004; ately processed on board. Gill, adductor muscle, mantle, foot, and vis- Kyuno et al., 2009; Yu et al., 2019). No significant genetic differentiation ceral mass (including the gut) were dissected using sterilized scissors was detected between the seep and vent populations, as indicated by and tweezers. After the dissection, the tissues were immediately frozen low level of genetic differentiation and extensive gene flow (Miyazaki in liquid nitrogen and then preserved at -80 °C. At the end of the cruise, et al., 2013; Shen et al., 2016), but the comparison of associated micro- all samples were transported to the laboratory on dry ice and stored at biota of these two species from two contrasting habitats is still limited -80 °C until subsequent analysis. so far. Moreover, as most previous studies investigated gill symbionts, information concerning the microbial community structure associated 2.2. DNA extraction, library construction, and amplicon sequencing with different tissues is yet to be reported. The Okinawa Trough (OT) is a typical back-arc basin that formed Total DNA of microbial community along with tissues was extracted from the subduction of the Philippine Sea Plate beneath the Eurasian from triplicate samples using the Fast DNA Spin Kit for Feces (MP Bio- Plate. At present, the OT is in the nascent stage of its evolution and is medicals, USA) and stored at -20 °C until PCR amplification. PCR still in the rifting stage prior to seafloor spreading (Sibuet et al., 1995). amplicons covering the hyper-variable V3V4 region of the 16S rRNA In this trough, a large section of the seawater is deeper than 1000 m, gene were amplified using the 341F-806R primer pair (5’-CCTACGGG and the thickness of sediments in the northern section is up to 8 km NGGCWGCAG-3’ and 5’-GGACTACHVGGGTATCTAAT-3’)modified to in- (Sibuet et al., 1987). The unique geographical features of the OT give clude adapters and unique barcodes for each sample. Samples was am- birth to a rare geological phenomenon in the world: the coexistence of plified in triplicate 25-μL reactions with the following cycling submarine cold seeps and active hydrothermal vents. According to in- parameters: 95 °C for 3 min, 30 cycles of 95 °C for 45 s, 50 °C for 60 s, complete statistics, more than 20 vent sites with different fluid temper- and 72 °C for 90 s, and a final elongation step at 72 °C for 10 min. atures have been discovered (Miyazaki et al., 2017) since the first Three negative controls were also processed along with the DNA extrac- discovery of a hydrothermal vent field in the OT in the 1980s tion and PCR amplification in order to figure out the possible reagent (Halbach et al., 1989). Similarly, pervasive seafloor cold seeps have and laboratory contamination (Salter et al., 2014). Following the

2 G. Lin, J. Lu, Z. Sun et al. Science of the Total Environment 796 (2021) 149046

Fig. 1. Sampling collection of Bathymodiolus mussels. The distance of Station S11 cold seeps and hydrothermal field of Minami-Ensei Knoll is about 50 km.

amplification, the triplicate products were first visualized on 1.5% aga- 2.4. Statistical analyses rose gels to assess amplification success, and then combined the tripli- cates together, quantified with the QuantiFluor dsDNA System Values were expressed as the means ± standard deviation. Data was (Promega, USA), pooled in equal amounts, and purified. The constructed statistically analyzed by one-way analysis of variance (ANOVA) library was sequenced on an Illumina MiSeq platform (Guangzhou Gene followed by LSD test with SPSS v21.0 to identify significant differences, Denovo Biotechnology Co., Ltd., China). and a p-value <0.05 was determined to be statistically significant.

2.3. Bioinformatic analyses 3. Results

Quality-controlled sequencing reads were paired into tags using 3.1. Microbial community diversity of two Bathymodiolus species from the FLASH v1.2.11 (Magoc and Salzberg, 2011). After trimming the low- adjacent cold seep and hydrothermal vent quality tags, filtering tags shorter than 300 bp, and chimera removal (Edgar et al., 2011), clean tags were obtained and clustered into opera- A total of 5,406,567 paired-end sequences were obtained after the tional taxonomic units (OTUs) at 97% identity using Uparse v9.2.64 quality control and were clustered into 3595 OTUs. OTUs in which 1% (Edgar, 2013). The representative sequences of OTUs, selected as the or greater of sequences were from the negative control library were most abundant ones, were aligned to the SILVA reference database considered likely background contamination. Therefore, 103 OTUs v132 (Quast et al., 2013) with a confidence threshold of 80% using a were removed from the dataset, resulting in a final curated dataset con- naïve Bayesian classifier (Wang et al., 2007). As a final quality control taining 3492 OTUs clustered from 5,369,147 sequences. Each library step, potential contaminant sequences were screened by removing contained an average of 91,002 ± 5475 sequences (SI Dataset S1). Rar- OTUs from the dataset wherein 1% or greater of its sequences were efaction curves for all samples approached saturation (SI Fig. S1), to- sourced from the negative control library (Doyle et al., 2018). The final gether with Good's Coverage values >99% (Fig. 2; SI Table S1), curated dataset was used for community diversity and composition indicating that the sequencing depth was sufficient and the remaining analyses by QIIME v1.7.0 and R packages “phyloseq” (McMurdie and unsampled diversity likely contained only rare taxa. With regard to Holmes, 2013) and “vegan” (Oksanen et al., 2019). The 16S rRNA se- community richness, Chao1 and ACE estimators did not change much quences of gammaproteobacterial methanogens and Escherichia coli among tissues (Chao1: 405.94–809.28; ACE: 426.61–774.46). The com- (outgroup), as well as epsilonbacteraeotal bacteria and Helicobacter munity diversity of the gill in seep mussels significantly diminished (p- pylori (outgroup) were downloaded from GenBank for phylogenetic value <0.01) compared with other tissues and their hydrothermal vent analyses. Sequence alignments were generated using ClustalW imple- counterparts, as indicated by Shannon (the gill of seep mussels: 1.07 ± mented in MEGA X v10.1.0 (Kumar et al., 2018) with default settings. 0.16; others: 1.49–3.54) and Inverse Simpson (the gill of seep mussels: Phylogenetic analyses were performed using the maximum likeli- 0.27 ± 0.05; others: 0.31–0.72) indices, whereas no significant differ- hood (ML) and Bayesian inference (BI) approaches as described in ences were observed between two species within the same environ- Xu et al. (2019). The ML analyses were conducted via MEGAX ment (Fig. 2;SITableS1). v10.1.0 (Kumar et al., 2018) using the General Time Reversible Bray-Curtis-based Non-metric multidimensional scaling (NMDS) model with gamma-distributed rate of evolution and invariant sites showed that gill samples from two environments clustered together (GTR + I + G), with a bootstrap replication number of 1000. The BI and segregated from other tissues, while other tissues showed divergence analyses were conducted via MrBayes v3.2.6 (Ronquist et al., 2012) between two environments (Fig. 3). Analysis of molecular variance using the same model. Four Markov chains were applied for 10 mil- (AMOVA) indicated significant differences in microbial community struc- lion generations and sample trees were taken every 1000 genera- ture among two environments (F(1, 57) = 103.232, p-value <0.001). The tions, with the first 25% discarded as the burn-in. Functional community structure of the same species from two different environ- prediction was analyzed by FAPROTAX (Functional Annotation of ments was significantly different (B. platifrons: F(1, 28) = 57.576, p-value Prokaryotic Taxa) (Louca et al., 2016) and PICRUSt2 (Phylogenetic <0.001; B. japonicus: F(1, 27) = 45.412, p-value <0.001). However, no no- Investigation of Communities by Reconstruction of Unobserved ticeably differences were observed between two different species from

States) (Douglas et al., 2020). the same environment (cold seep: F(1, 27) = 0.548, p-value = 0.507;

3 G. Lin, J. Lu, Z. Sun et al. Science of the Total Environment 796 (2021) 149046

Good's Coverage Chao1 Shannon

Gill Gill Gill a a100% a 900 4

a a ef 99.5% 3 ab600a cde a b a bcdef a f Visceral mass Visceralb mass a Visceralf mass ab b 99% 2 d ef ab

300 a a a a

5% c 1 de

98. c

ab b ab ab a abcde ab a 0 0 98% a ab abcde muscle

ab r a cd de

e abc a f a ab ab ab cdef Foot Foot ab Foot b a ab abc ab d a ab ab abcde cde cdef Adducto Adductor muscle abcde ab Adductor muscle a a

a a a Mantle Mantle Mantle

Observed OTUs ACE Inverse Simpson

Gill Gill Gill 800 900 0.8 f

ef 600 ef 0.6 a600 a a ab a ef Visceralb mass Visceral mass a Visceral massef a a a 400 ab 0.4a a a a a 300

200 2 ef

a 0. b a

a a abc bcde a a

0 0 a 0 ab def

a ab r muscle a r muscle a a f or muscle ef e a ab d c a a a abc a d Foot a a Foot Foot a a ab ef a a a Adducto Adduct cdef f Adducto cdef cde ef

Mantle Mantle Mantle

cold seep B. platifrons cold seep B. japonicus hydrothermal vent B. platifrons hydrothermal vent B. japonicus

Fig. 2. Alpha diversity of microbial community in different tissues of B. platifrons and B. japonicus from the adjacent cold seep and hydrothermal vent environments. Groups with different letters were significantly different (p-value <0.05).

hydrothermal vent: F(1, 28) = 0.653, p-value = 0.601). Analogously, at the higher than that of Pseudomonadaceae, in the gill of B. platifrons and tissue level, the community structure of adductor muscle, mantle, foot, B. japonicus, respectively. On the contrary, the opposite was observed and visceral mass showed significant differences between environments in the other four tissues; the relative abundances of Pseudomonadaceae rather than species. Notably, the community structure of the gill were sig- were about 1.4 to 5.7 times higher than that of Methylomonaceae in ad- nificantly different from that of other tissues (seep B. platifrons: F(1,13) = ductor muscle and mantle, and this fold change values increased to 37.563, p-value <0.001; seep B. japonicus: F(1,12) = 35.854, p-value 9–20 in foot and visceral mass of two species. In vent mussels, Entero- <0.001; vent B. platifrons: F(1, 13) = 4.034, p-value = 0.024; vent bacteriaceae showed predominance in all tissues of two species, B. japonicus: F(1, 13) = 19.834, p-value = 0.002), while the communities which was 3- to 185-fold more abundant than Methylomonaceae, of gill samples were more similar even across different environments (the gill of seep B. platifrons vs. the gill of vent B. platifrons: F(1, 4) = stress = 0.031 26.630, p-value = 0.083; the gill of seep B. japonicus vs. the gill of vent

B. japonicus: F(1, 4) = 22.818, p-value = 0.089). Among all the compari- sons between gill samples, only the differences between the gill of seep 0.2

B. japonicus and the gill of vent B. platifrons were significant (F(1, 4) = 38.562, p-value = 0.016).

3.2. Microbial community composition of two Bathymodiolus species from 0.0 the adjacent cold seep and hydrothermal vent NMDS2

Gill Among various lineages, Gammaproteobacteria was overwhelm- Adductor muscle ingly the most abundant across all the tissues (Fig. 4). A total of 552 -0.2 Mantle OTUs affiliated with Gammaproteobacteria together comprised 83.99% Foot of all sequences, with the highest relative abundance (97.19%) detected Visceral mass in the gill of seep B. japonicus and the lowest (61.02%) in visceral mass of -0.2 0.0 0.2 vent B. japonicus. Nevertheless, the predominated family member of NMDS1 Gammaproteobacteria varied between two environments, namely, cold seep B. platifrons hydrothermal vent B. platifrons Methylomonaceae and Pseudomonadaceae in seep mussels while En- cold seep B. japonicus hydrothermal vent B. japonicus terobacteriaceae and Methylomonaceae in vent ones. In seep mussels, the ratio of Methylomonaceae to Pseudomonadaceae appeared to be af- Fig. 3. NMDS plot of the shifts in the microbial community structure observed in different fected by tissue types. The relative abundances of Methylomonaceae tissues of B. platifrons and B. japonicus from the adjacent cold seep and hydrothermal vent were 85.84 ± 2.94% and 87.18 ± 5.04%, which was 46- and 22-fold environments.

4 G. Lin, J. Lu, Z. Sun et al. Science of the Total Environment 796 (2021) 149046 with only one exception observed. In the gill of vent B. japonicus,therel- Microbacteriaceae), and OTU8 (genus Mesorhizobium)wereobservedin ative abundances of Enterobacteriaceae (38.41 ± 13.12%) and all tissues of hydrothermal vent individuals (relative abundances: Methylomonaceae (48.52 ± 12.53%) were comparable. 2.23–14.91%), in contrast to their rarity in cold seep individuals (relative Except for the Gammaproteobacteria, another two classes affiliated abundances: 0–0.47%). It's also noteworthy that two OTUs were more with Proteobacteria, Alphaproteobacteria and Deltaproteobacteria, abundant in visceral mass than in other tissues. The relative abundances were also observed in all tissues (Fig. 4). The relative abundances of of OTU6 (class Mollicutes) were 11.94–12.72% in visceral mass of vent Alphaproteobacteria were higher in vent mussels (3.16–7.06%) than in mussels, and the relative abundances of OTU12 (genus Sulfurovum) seep ones (0.15–2.08%), while Deltaproteobacteria remained static were 2.04–2.37% in visceral mass of vent mussels and seep and lower relative abundances (0.04–2.34%) in two environments B. japonicus, while in other tissues the abundances of these two OTUs with highest value detected in the gill of vent B. platifrons.Several were no more than 2.22%. phyla also showed heterogeneity among environments and/or tissues. For example, phyla Acidobacteria, Actinobacteria, and Bacteroidetes 3.3. Putative functions of microbial community of two Bathymodiolus were more abundant in vent mussels than in seep mussels, while species from the adjacent cold seep and hydrothermal vent phylum Epsilonbacteraeota were more abundant in seep ones. Planctomycetes and Tenericutes were two phyla which were detected Functional prediction through PICRUSt2 demonstrated a distinguishing more frequently in visceral mass than in other tissues. cluster of the gill, characterized by the large count number of putative en- The top 15 most abundant OTUs contained about 90% of all se- zymes involved in methane oxidation (count: 8858.27–37,368.61), ATP quences, exhibiting relatively low diversity of microbial communities metabolism (count: 9467.05–37,537.26), H4MPT (tetrahydrometha- of both B. platifrons and B. japonicus from two environments (Fig. 5;SI nopterin) pathway (count: 8928.09–74,829.29), and RuMP (ribulose Dataset S1). These OTUs drove largely the shifts of microbial community monophosphate) pathway (count: 8880.79–74,733.83) (Fig. 6a). Consis- composition at higher taxonomy level. About 85% of the total commu- tently, possible functional groups in gill-associated bacterial community nity represented by a single OTU (OTU1, family Methylomonaceae) in were characterized by methanotrophy, methylotrophy, and hydrocarbon the gill of seep mussels (B. platifrons: 85.73 ± 2.97%; B. japonicus: degradation, with relative abundances ranging from 10.96% to 23.67% re- 84.95 ± 3.66%), resulting in their low community diversity (Fig. 2;SI vealed by FAPROTAX analyses (Fig. 6b). In addition, putative enzymes Table S1). As for the gill of vent mussels, the relative abundances of and function groups relevant to sulfur, nitrogen, and hydrogen cycles OTU1 decreased but still accounted for 17.39–43.26% of all sequences. patchily distributed in different tissue of two mussel species from two hab- Similarly, two Campylobacteria OTUs (OTU5 and OTU15, family itats. For example, hypD (hydrogenase expression/formation protein) in- Helicobacteraceae) also exhibited higher relative abundances in the volved in hydrogen metabolism and dmsA (dimethyl sulfoxide reductase gill of seep B. platifrons and B. japonicus from two environments, relative subunit A) involved in sulfur metabolism were predicted to be more abun- to other tissue types. In addition, OTU10 (genus Methyloprofundus) dant in hydrothermal vent mussels, whereas sqr (sulfide: quinone oxido- representing a “Gammaproteobacteria within Bathymodiolus japonicus reductase) and cysNC (bifunctional enzyme CysN/CysC) involved in sulfur methanotrophic gill symbiont” was detected in the gill of not only metabolism were predicted to be more abundant in cold seep mussels. In B. japonicus from two environments but also vent B. platifrons, with rel- comparison, several putative enzymes showed abundant in almost all tis- ative abundance spanning between 1.78% and 4.16%, while in other tis- sues, such as pgd (6-phosphogluconate dehydrogenase), frdB (succinate sues the abundances were no more than 0.39%. Some OTUs showed dehydrogenase / fumarate reductase, iron-sulfur subunit), eno (enolase), abundant in the four tissues (adductor muscle, mantle, foot, and visceral cysZ (sulfate transporter CysZ protein), and nrtA (nitrate/nitrite transport mass) other than the gill in cold seep individuals, such as OTU3 (genus system substrate-binding protein) (Fig. 6). Pseudomonas), OTU13 (genus Ralstonia), OTU14 (genus Ralstonia), and OTU16 (genus Burkholderia-Caballeronia-Paraburkholderia). These 4. Discussion OTUs also displayed low relative abundances (< 0.39%) in all tissues of vent mussels. Apart from the 1.35-fold higher abundance of OTU1 In this study, we characterized tissue-associated microbial than OTU2 (family Enterobacteriaceae) in the gill of vent B.japonicus community diversity, composition, and metabolic potential of two which was in line with the observations of community composition at Bathymodiolus species collected from the adjacent cold seep and hydro- family level, OTU2 was the overwhelmingly dominant OTU in other tis- thermal vent environments. sues of vent mussels, comprising 47.73–67.90% of the total microbial The gill serves as an interface with the external marine environment community. OTU4 (family Enterobacteriaceae), OTU7 (family in a wide range of mussel species (Lee and Childress, 1994; Riou et al.,

Minor Members cold seep B. platifrons cold seep B. japonicus hydrothermal vent B. platifrons hydrothermal vent B. japonicus Unclassified Bacteria 100 Phylum Acidobacteria Actinobacteria Bacteroidetes 80 Epsilonbacteraeota Firmicutes Patescibacteria

60 Planctomycetes Tenericutes Verrucomicrobia Class (Proteobacteria) 40 Alphaproteobacteria Deltaproteobacteria

Relative abundance (%) Family (Gammaproteobacteria) Burkholderiaceae 20 Endozoicomonadaceae Enterobacteriaceae Methylomonaceae Neisseriaceae 0 Pseudomonadaceae GL AM MT FT VM GL AM MT FT VM GL AM MT FT VM GL AM MT FT VM

Fig. 4. Relative abundances of the various microbial lineages observed in different tissues of B. platifrons and B. japonicus from the adjacent cold seep and hydrothermal vent environments. Each bar is the average of triplicate treatments. Taxa with relative abundances <1% are combined into the minor members category. GL, gill; AM, adductor muscle; MT, mantle; FT, foot; VM, visceral mass.

5 .Ln .L,Z u tal. et Sun Z. Lu, J. Lin, G. 0.5 Relative abundance (%) 1.5 Relative abundance (%) Relative abundance (%) Relative abundance (%) Relative abundance (%) 30 60 90 12 12 18 0 3 6 9 0 1 0 0 4 8 0 6 GL GL GL GL GL MM FT MT AM MMT AM MMT AM MMT AM MMT AM FT FT FT FT VM VM VM VM VM GL GL GL GL GL MM FT MT AM MMT AM MM FT MT AM MM FT MT AM MMT AM FT FT VM VM VM VM VM GL GL GL GL GL Enterobacteriaceae Microbacteriaceae Methylomonaceae MMT AM MMT AM MM FT MT AM MM FT MT AM MM FT MT AM Endozoicomonas FT FT VM VM VM VM VM GL GL GL GL GL Ralstonia MMT AM MMT AM OTU14 MMT AM MMT AM MMT AM OTU11 OTU1 OTU4 OTU7 FT FT FT FT FT VM VM VM VM VM 30 60 90 0 0 3 6 9 0 1 2 3 0 1 2 3 0 2 4 6 GL GL GL GL GL MMT AM MMT AM MMT AM FT MT AM MMT AM FT FT FT FT VM VM VM VM VM GL GL GL GL GL MMT AM MMT AM MMT AM MT AM MMT AM 6 FT FT FT FT FT VM VM VM VM VM GL GL GL GL GL Enterobacteriaceae Helicobacteraceae Helicobacteraceae MMT AM MMT AM MMT AM MT AM MMT AM Mesorhizobium FT FT FT FT FT VM VM Sulfurovum VM VM VM GL GL GL GL GL OTU12 OTU15 MMT AM MMT AM MM FT MT AM FT MT AM MMT AM OTU2 OTU5 OTU8 FT FT FT VM VM VM VM VM 0.5 1.5 0.5 1.5 10 15 60 30 90 1 0 0 5 0 0 2 4 6 0 1 GL GL GL GL GL MMT AM MMT AM MMT AM MMT AM MMT AM FT FT FT FT FT cec fteTtlEvrnet76(01 149046 (2021) 796 Environment Total the of Science VM VM VM VM VM GL GL GL GL GL MMT AM MMT AM MMT AM MMT AM MM FT MT AM Burkholderia-Caballeronia- FT FT FT FT VM VM VM VM VM GL GL GL GL GL Methyloprofundus MMT AM MMT AM MMT AM MMT AM MM FT MT AM Paraburkholderia FT FT FT FT Pseudomonas VM VM VM VM VM Mollicutes GL GL GL GL GL Ralstonia OTU16 MMT AM OTU10 OTU13 MMT AM MMT AM MMT AM MM FT MT AM OTU3 OTU6 FT FT FT FT VM VM VM VM VM G. Lin, J. Lu, Z. Sun et al. Science of the Total Environment 796 (2021) 149046

(a)

log10(Count) K10944 pmoA-amoA

K10945 pmoB-amoB methane oxidation 4.5 K10946 pmoC-amoC 4 K02117 atpA K02118 atpB ATP metabolism 3.5

K02120 atpD 3 K04199 mch 2.5 K00672 ftr H4MPT pathway K14028 mdh1 K08093 hxlA K08094 hxlB RuMP pathway K13831 hps-phi K06045 shc K00033 pgd K01674 cah K00240 frdB Carbohydrate K01689 eno transport and metabolism K01610 pckA K00850 pfkA K00801 fdft1 Cholesterol biosynthesis K04654 hypD Hydrogen metabolism K17222 soxA K06203 cysZ K17218 sqr Sulfur metabolism K00955 cysNC K07306 dmsA K04561 norB K15576 nrtA nitrogen metabolism K00261 gdhA CS_P_GL CS_J_GLHV_J_GL HV_P_GL CS_P_AM CS_J_FT CS_J_MT CS_P_MT CS_P_FTCS_J_AM CS_J_VM CS_P_VM HV_P_AM HV_J_AM HV_J_MT HV_P_FT HV_J_FT HV_P_MT HV_P_VM HV_J_VM

cold seep B. platifrons hydrothermal vent B. platifrons (b) cold seep B. japonicus hydrothermal vent B. japonicus Gill Adductor muscle Mantle Foot Visceral mass

Relative Abundance (%) methanotrophy methylotrophy 20 hydrocarbon degradation aromatic compound degradation 15

aliphatic non-methane hydrocarbon degradation 10 respiration of sulfur compounds sulfate respiration 5 sulfite respiration 0 thiosulfate respiration dark sulfide oxidation / dark oxidation of sulfur compounds nitrate respiration nitrate reduction nitrogen fixation nitrification denitrification dark hydrogen oxidation HV_P_GL HV_J_GLCS_P_GL CS_J_GL HV_P_MT HV_P_AM HV_J_FT HV_P_FTHV_J_AM HV_J_MT CS_J_MT CS_J_FT CS_P_AM CS_J_AM CS_P_MTCS_P_FT CS_J_VM CS_P_VM HV_P_VM HV_J_VM

Fig. 6. Functional profiles of microbial community in different tissues of B. platifrons and B. japonicus from the adjacent cold seep and hydrothermal vent environments predicted through (a) PICRUSt2 and (b) FAPROTAX. GL, gill; AM, adductor muscle; MT, mantle; FT, foot; VM, visceral mass.

2008; Musella et al., 2020). Our results demonstrated high abundance of sequences of known methanotrophic symbionts of deep-sea mussels in gammaproteobacterial OTUs in the gill, which parallels previous obser- West Pacific Ocean. This clade is separated from those associated with vations in deep-sea mussels collected from the South China Sea (Xu deep-sea mussel species found in other locations such as the Gulf of et al., 2019; Sun et al., 2020). The gill of seep and vent mussels were Mexico and the Mid-Atlantic Ridge. Free-living type I methane oxidizers all dominant by a single OTU affiliated with family Methylomonaceae in the genera Methylobacter, Methylomicrobium,andMethylomonas (OTU1). Phylogenetic analysis demonstrated that OTU1 represents a form a sister group to the symbiotic methanotrophs (Fig. 7a). The rela- bacterial species closely related to cold seep Gigantidas haimaensis n. tive abundance of OTU1 was about 85% in the gill of seep mussels, com- sp. associated bacterium (Xu et al., 2019) within a single monophyletic parable with the results in other seep mussel species (Duperron et al., group which clearly falls within a larger clade only containing 16S rRNA 2007; Xu et al., 2019), which was also the reason why gill microbial

Fig. 5. Relative abundances of the 15 most abundant OTUs observed in different tissues of B. platifrons and B. japonicus from the adjacent cold seep and hydrothermal vent environments. Each bar is the average of triplicate treatments. Color legend is the same as Fig. 2 and Fig. 3: green is cold seep B. platifrons, blue is cold seep B. japonicus, pink is hydrothermal vent B. platifrons, and red is hydrothermal vent B. japonicus. Tick marks on the x-axes are in the same order as those in Fig. 4: gill (GL), adductor muscle (AM), mantle (MT), foot (FT), and visceral mass (VM). No data presented reflects the absence of the OTU in that tissue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

7 G. Lin, J. Lu, Z. Sun et al. Science of the Total Environment 796 (2021) 149046

(a) B. brooksi symbiont (Psychromonas) in the GoM (JF969168) Methylobacter psychrophilus (AF152597) Methylomonas rubra (M95662) Methylomonas sp. (M95658) 77 / 0.989 free-living type I methane oxidizers 97 / 0.992 Methylobacter luteus (X72772) Methylomicrobium album BG8 (X72777) 92 / 0.799 64 / 0.872 Methylomicrobium pelagicum (X72775) 87 / 0.891 Cold seep Gigantidas haimaensis n. sp. associated bacterium (MK534991) OTU1 Methylomonaceae 65 / - 97 / 0.930 Cold seep B. platifrons associated OTU (MT052681) 70 / - deep-sea mussel associated 51 / - Okinawa Trough B. platifrons symbiont (AB250696) Hydrothermal vent B. platifrons symbiont (AB036710) methanotrophs in West Pacific Ocean 94 / 0.973 Hydrothermal vent B. japonicus symbiont (AB036711) 92 / 0.851 OTU10 Methyloprofundus Methane seep B. sp. endosymbiont in southeast Atlantic (AJ745717) 100 / 0.950 97 / 0.709 Cold seep B. heckerae endosymbiont in the GoM (M236325) 99 / 0.999 Mud volcano B. mauritanicus symbiont in Atlantic Ocean (HE963013) deep-sea mussel associated Louisiana Slope mussel Ia symbiont (U05595) 99 / 1.000 52 / - Cold seep B. childressi endosymbiont in the GoM (AM236329) methanotrophs in other locations 53 / - Hydrothermal vent B. sp. endosymbiont in the MAR (JQ844775) 50 / - Hydrothermal vent B. sp. endosymbiont in the MAR (JQ844776) B. hirtus symbiont in west Pacific (AB250698) Hydrothermal vent B. azoricus endosymbiont from Lucky Strike in the MAR (AM083953) 0.01 64 / - Hydrothermal vent B. puteoserpentis endosymbiont from Logatchev in the MAR (AM083963) 99 / 1.000 Hydrothermal vent B. azoricus endosymbiont from Rainbow in the MAR (AM083954) deep-sea mussel associated 84 / 0.915 Hydrothermal vent mussel symbiont in the MAR (U29164) Hydrothermal vent B. azoricus endosymbiont from Menez Gwen in the MAR (AM083967) methanotrophs in other locations 89 / 0.925 Cold seep B. brooksi symbiont in the GoM (JF969169) 75 / 0.890 74 / - Cold seep Idas sp. endosymbiont in the eastern Mediterranean (AM402955) (b) Methyloprofundus sedimenti from marine sediment (KF484906) Campylobacter jejuni (NR_117760) Kiwa puravida (Yeti crab) associated epsilonproteobacterium (JN255994) 80 / 0.994 Hydrothermal vent gastropod associated bacterium in Indian Ocean (AY531574) 82 / 0.940 Sulfurovum lithotrophicum from hydrothermal vent sediment (NR_024802) 81 / 0.807 Hydrothermal vent Riftia pachyptila (tubeworm) associated Sulfurovum riftiae (KP896689) 66 / - Sulfurovum aggregans from a hydrothermal vent chimney (AB889689) Sulfurovum sp. from the hydrothermal field (LC322101) 67 / 0.914 OTU12 Sulfurovum 63 / 0.902 91 / 1.000 Hydrothermal vent gastropod gill endosymbiont in Indian Ocean (AB205405) 98 / 0.997 Uncultured Sulfurovum sp. from a cold seep in the SCS (MT052679.1) 55 / 0.918 55 / - Hydrothermal vent Kiwa sp. (crab) bacterium episymbiont from East Scotia Ridge Antarctica (KF438966) Hydrothermal vent Rimicaris exoculata (shrimp) ectosymbiont (FN658695) 98 / 1.000 Acropora cervicornis (coral) associated uncultured bacterium (GU117948) 92 / 1.000 Muricea elongata (coral) associated uncultured Helicobacter sp. (DQ917867) 95 / 0.999 OTU15 Helicobacteraceae Cold seep Thyasira flexuosa (bivalve) associated bacterium in the Eastern Mediterranean (FN600361) 94/ 1.000 88 / 0.929 B. sp associated uncultured bacterium (KU573880) B. sp associated uncultured bacterium (KU573879) 96 / 0.995 Cold seep Gigantidas haimaensis n. sp. associated uncultured bacterium in the SCS (MK534992) 61 / 0.977 95 / 1.000 OTU5 Helicobacteraceae Okinawa Trough B. platifrons uncultured gill symbiont (AB250697) 61 / - deep-sea mussel associated 60 / - Cold seep B. platifrons associated OTU in the SCS (MT052682) 79 / 0.895 B. manusensis associated uncultured bacterium from Fenway, Lau Basin (KU573870) 0.01 epsilonbacteraeota 51 / - B. azoricus associated uncultured bacterium from Lucky Strike in the MAR (KU573852) 51 / - B. azoricus associated uncultured bacterium from Rainbow in the MAR (KU573850) B. sp. associated uncultured bacterium from Lilliputh in the MAR (KU644658) B. hildressi associated uncultured bacterium in the GoM (KU573856) B. sp. associated uncultured bacterium in the MAR(KU644660) B. mauritanicus assoicated uncultured bacterium from Milano, Barbados accretionary complex (KU573874) B. azoricus assocaited uncultured bacterium from Menez Gwen in the MAR (KU573846)

Fig. 7. Phylogenetic relationships based on representative sequences of OTUs and 16S rRNA gene sequences retrieved from (a) gammaproteobacterial methanotrophs (465 bp) and (b) epsilonbacteraeotal bacteria (441 bp) based on the ML topology. OTUs in this study are marked in bold and shaded in grey. Bootstrap values inferred from the ML analysis and posterior probabilities inferred from the BI analysis are shown at nodes. Dashes (-) indicate posterior probabilities below 0.70. GoM, Gulf of Mexico; MAR, Mid-Atlantic Ridge; SCS, South China Sea. community diversity of cold seep mussels was low. Intriguingly, the rel- B. japonicus and the two vent mussel species. Our results confirmed ative abundance of OTU1 reduced by half in vent mussels, suggesting that OTU10 is closely related to symbiont of hydrothermal vent the host adaptation to environmental changes through dynamically B. japonicus (Fig. 7a). These two symbiotic OTUs were also observed in changing the abundances of methanotrophs. The abundances of chemo- other tissues, but with lower relative abundances. The probable reason synthetic symbionts in deep-sea mussels have been reported to be envi- was that the symbiotic bacteria are widespread in every tissue during ronmentally dependent (Bergquist et al., 2004), which can contribute to the early developmental stage and then specifically colonize the gill the optimization of energy yield for the symbiotic system (Duperron (Wentrup et al., 2013). et al., 2011). To be specific, methanotrophic symbioses is influenced The distinct pattern of microbial community structure in mussel gill by the concentration of methane supplied by the seep or vent environ- potentially reflected its tissue-peculiar functions. Predicted functional ments (Petersen and Dubilier, 2009; Sun et al., 2017b). Nevertheless, profiles displayed a large proportion of encoding enzymes involved in the relative abundances showed no obvious differences between two carbon and energy metabolism. Several putative encoding enzymes host species from the same habitats, in agreement with the previous ob- including pmoA/B/C (methane/ammonia monooxygenase subunit A/ servations of Bathymodiolus mussels (Duperron et al., 2006). However, B/C), mch (methenyltetrahydromethanopterin cyclohydrolase), ftr the variability of the relative abundance and the gene expression of (formylmethanofuran-tetrahydromethanopterin N-formyltransferase), mussel symbionts (Guezi et al., 2014) should be further evidenced mdh1 (methanol dehydrogenase subunit 1), hxlA (3-hexulose-6-phos- using larger number of samples. In addition, another OTU affiliated phate synthase), hxlB (6-phospho-3-hexuloisomerase), hps-phi with Methylomonaceae (OTU10, genus Methyloprofundus) accounted (3-hexulose-6-phosphate synthase / 6-phospho-3-hexuloisomerase), for about 2–4% of total abundance of gill microbial community in seep and shc (squalene-hopene/tetraprenyl-beta-curcumene cyclase) were

8 G. Lin, J. Lu, Z. Sun et al. Science of the Total Environment 796 (2021) 149046 abundantly presented in the gill. These predicted enzymes were associ- affiliated with phylum Actinobacteria and genus Mesorhizobium affili- ated with methane oxidation, H4MPT (tetrahydromethanopterin) path- ated with phylum Proteobacteria. The existence of most of these groups way, and RuMP (ribulose monophosphate) pathway, which have been has been reported from hydrothermal fluids and/or gas hydrate sedi- defined as the dominated metabolism processes in gill symbionts of ments and get involved in element cycling (Orcutt et al., 2011; seep and vent mussels (Ponnudurai et al., 2017a; Wang et al., 2021), Fortunato and Huber, 2016). Some members of Ralstonia are capable further backing that the potential functions of gill microbiota of of hydrogen oxidization, iron reduction, and denitrification (Marchesi Bathymodiolus mussels from two environments both included energy et al., 2001; Lin et al., 2007), while Burkholderia has the potential for and biomass generation as well as formaldehyde detoxification. Mean- methylotrophy, nitrogen reduction, and hydrocarbon metabolism while, high abundances of a predicted enzyme involved in cholesterol (Mills et al., 2012; Pérez-Pantoja et al., 2012). Pseudomonadaceae and biosynthesis, fdft1 (farnesyl-diphosphate farnesyltransferase), were Enterobacteriaceae families showed prevalence in deep-sea sediments observed in the gill of mussels collected from both environments. This cultured with hydrocarbons (Moreno-Ulloa et al., 2020), thus may get putative enzyme can synthesize sterol intermediate from the product involved in hydrocarbon degradation. Enterobacteriaceae has hydroge- of methane oxidation, which is dependent on gill methanotrophic sym- nase activity (Krasna, 1980)andMesorhizobium has denitrification abil- bionts (Takishita et al., 2017). FAPROTAX again evidenced that ity (Yu et al., 2018). In addition, it is also reasonable that tissues were methanotrophy was predicted as the dominant process in the gill, infected by pathogens because Pseudomonadaceae, Enterobacteriaceae, displaying consistency with previous study that methanotrophy, and Burkholderiaceae (Ralstonia and Burkholderia)allcontainsome methylotrophy, and hydrocarbon degradation were dominant function pathogenic taxa. Therefore, the interactions of these bacterial groups groups for cold seep mussel Gigantidas haimaensis (Ling et al., 2020). with different tissues of deep-sea mussels required further exploration. Moreover, functions involved in nitrogen and sulfur cycles were also Although the enlarged gill and reduced digestive tract (i.e. the short predicted regardless of relatively low abundance. The stable isotopic and straight gut) were observed, it remains unclear that whether measurements have unraveled the capacity of B. platifrons and B. platifrons and B. japonicus retain the ability to ingest suspended parti- B. japonicus for assimilating seawater sulfate‑sulfur as well as isotopi- cles and assimilate particulate organic matter through filter-feeding or cally light nitrate or ammonium (Yamanaka et al., 2000; Feng et al., not, as described in some other deep-sea mussel species (Page et al., 2015). Meta-proteomics of the B. platifrons gill also provided evidence 1990, 1991; Fujiwara et al., 1998). Actually, we found two OTUs solely to support the involvement of pathways such as assimilatory sulfate re- abundant in the visceral mass. OTU6 (class Mollicutes) was observed duction and nitrogen metabolism in Bathymodiolus (Sun et al., 2017a). in visceral mass of the two vent mussel species with relative abun- The expression patterns of the aforementioned predicted genes could dances of more than 11%. Mollicutes affiliated with phylum Tenericutes be further demonstrated through metatranscriptome sequencing is one of the main classes in the gut microbial communities of several approach. deep-sea organisms such as shrimps (Durand et al., 2015) and amphi- Phylum Epsilonbacteraeota (reclassification of class Epsilonpro- pods (Cheng et al., 2019). In addition, the relative abundances of teobacteria, Waite et al., 2017), known for chemolithotrophy in various OTU12 (genus Sulfurovum) were more than 2% in visceral mass of marine environments including hydrothermal vents, contains symbiotic seep B. japonicus and the two vent mussel species. OTU12 belonged to assemblages of various invertebrates such as worms, shrimps, crabs, a large clade which includes hydrothermal animals associated bacte- and snails (Campbell et al., 2006; Goffredi, 2010; Li et al., 2020). Our re- rium as well as four environmental sequences (Fig. 7b). Sulfur- sults identified two OTUs affiliated with a host-associated family oxidizing genus Sulfurovum, widely distributed in deep-sea sediments, Helicobacteraceae. OTU5 and OTU15 showed more abundant in the hydrothermal vents, and hydrocarbon-rich environments (Campbell gill than in other tissues in both two vent mussel species and seep et al., 2006; Teske et al., 2021), dominates the microbial communities B. platifrons. OTU5 was phylogenetically clustered into the group of bac- of intestine and gill of Shinkaia crosnieri collected from the hydrother- teria which was specifically associated with deep-sea mussels, and mal vent in the Okinawa Trough and a cold seep in South China Sea, OTU15 was placed into a well-supported monophyletic clade only con- and may play a vital biogeochemical role in the carbon, sulfur, and nitro- taining the cold seep bivalve Thyasira flexuosa associated bacterium gen cycles (Zhang et al., 2018; Sun et al., 2020). These observations sug- which is a sister group to sequences of coral associated bacterium gested a specific association between Sulfurovum and deep-sea (Fig. 7b). Epsilonbacteraeota associates as gill filamentous epibionts invertebrates including crabs and mussels. with deep-sea mussels, especially with mussels that host only In conclusion, tissue-associated microbial community structure of methane-oxidizing symbionts (Assié et al., 2016; Coykendall et al., two Bathymodiolus species (B. platifrons and B. japonicus)collected 2019). The loss of the gammaproteobacterial sulfur-oxidizing symbi- from the adjacent cold seep and hydrothermal vent were characterized. onts in B. platifrons and B. japonicus may provide a new ecological In this study, environmental type (seep or vent) is not the primary fac- niche for sulfur-oxidizing Epsilonbacteraeota, so that these epibionts tor determining the gill symbiotic type of B. platifrons and B. japonicus, can access reduced sulfur compounds from fluids and transfer nutrients but could influence the abundance of gill symbionts as well as tissue- to the host (Zbinden et al., 2015; Assié et al., 2016). Therefore, even associated microbial community structure. Microbial community of though with relatively low abundance (< 6%) on host species compared two mussel species from the same sampling site shared a series of sim- with gammaproteobacterial symbionts, Epsilonbacteraeota may also ilarities, while microbial community in different tissue types showed play vital roles in gill symbiosis of deep-sea mussels. More bacterial structural variance. Functional predictions revealed that tissue- symbionts, including different symbioses involved in sulfide- and associated bacterial community get involved in element cycling, with methane-oxidizing or even novel types of symbionts, are being de- methane oxidation as the major putative functions specifically for gill- scribed (Duperron et al., 2007; Raggi et al., 2013; Rubin-Blum et al., associated community. This study can extend the understanding on 2017; Ansorge et al., 2019), potentially due to the adaptation to variable the characteristics of tissue-associated microbiota of two mussel species local environments, suggesting that the taxonomic diversity and meta- from the adjacent cold seep and hydrothermal vent environments. Fu- bolic versatility of deep-sea mussel symbionts is underestimated. ture study can focus on the further explanation of the possible mecha- Several other groups showed distinct patterns between two envi- nisms shaping tissue-associated microbiota of deep-sea mussels by ronments with no species specificity. Pseudomonas displayed predomi- combining microbial information with environmental geochemical nance in the four tissues other than the gill in seep mussels, identical data. Meanwhile, with the aim of illustrating the dynamic profiles of to the distribution of Ralstonia and Burkholderia-Caballeronia- the environmental adaptation of deep-sea mussels and the tissue- Paraburkholderia both affiliated with family Burkholderiaceae. In com- associated microbiota, the incubation experiments supplied with differ- parison, gammaproteobacterial Enterobacteriaceae were abundant in ent substrates and metagenome/metatranscriptome sequencing strat- all tissues of vent mussels, followed by family Microbacteriaceae egy can be jointly employed.

9 G. Lin, J. Lu, Z. Sun et al. Science of the Total Environment 796 (2021) 149046

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