Sampling and preparation of gastropods

Individuals of A. marisindica (kAlv and eAlv populations) were collected with a suction pump sampler equipped on the Shinkai6500 from representative habitats of the Kairei and Edmond fields, respectively. As summarized in Fig. S2 of the Supplemental materials, some of the gastropod individuals were collected and then were fixed in situ by using RNA later (25 mM sodium citrate, pH 5.2, 10 mM EDTA, 0.7 kg/L ammonium sulfate) [39]. This method (without the in situ homogenization of the gastropod) did not assure appropriate fixation of the endosymbiont’s intracellular RNA by potentially incomplete infiltration of RNA later solution into the tissue, while it was confirmed that the gastropod individuals after this fixation process provided greater abundances of quantification for the 16S rRNA and other functional gene transcripts from the gill endosymbiotic cells than the individuals without the in situ fixation in the quantitative PCR (qPCR) analyses described later. After recovery onboard, most of the individuals were reared in tanks containing fresh surface seawater previously cooled at 4 °C for more than 12 h. Other individuals were dissected and the gill tissues (and some of the mantle tissues) were subsampled and stored at −80 °C and 4 °C. The frozen gill and mantle tissues were used for DNA and protein analyses.

To measure H₂- and sulfide-consumption activity, both living A. marisindica individuals and dissected whole gill tissues (from individuals representing 3.5–4.0 cm shell width and 5.0–6.0 cm shell length, and within 12 h of onboard recovery) were washed twice with chilled surface seawater (previously passed through a 0.2 μm sterile filter) and incubated with substrates of either H₂ or sulfide. In addition, the gill tissues dissected from the in situ fixed individuals were preserved into RNAprotect Bacteria Reagent (Qiagen, Valencia, CA, USA) and then stored at −80 °C for the subsequent transcript and ISH analyses. The procedure of this study is illustrated in Fig. 1c.