Science of the Total Environment 796 (2021) 149046 Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv 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,