Data set of DNA sequences of Bathymodiolus azoricus juveniles attached to the shell of Protolira valvatoides.

In the present study, we report the observation of post-larval juveniles of B. azoricus attached to the shell of different living gastropod species, and more particularly the species Protolira valvatoides—a previously undocumented behavior, except for a brief mention page 152 in Desbruyères et al., (2006). Using stereo and scanning electron microscopy, along with molecular identification, we confirmed the identity of epibiont and searched for mussel larvae settlement in older samples. During the Momarsat cruise in August 2024 (Matabos & Sarradin, 2024), three active hydrothermal vent sites—Helene (dive 864), Vazydon (dive 863), and Y3 (dive 863)—within the Lucky Strike Hydrothermal Field (LSHF, Fig.1 ; 37°17′N, 32°16′W) were explored using the ROV Victor6000 deployed from the RV Atalante. At each site, a single sample of mussel-bed and associated fauna was collected using the ROV's robotic arm for alpha diversity assessment within B. azoricus aggregations. Samples were processed onboard through serial sieving (1 mm, 300 µm, 32 µm mesh sizes). Organisms retained in the > 1 mm fraction were counted and identified to broad taxonomic groups using a stereomicroscope. Gastropods exhibiting attached B. azoricus post-larvae were individually isolated for subsequent morphological and genetic analysis. Nine mussel (M11 to M19) juveniles were carefully removed from the P. valvatoides shells under the stereomicroscope. DNA identification was performed targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) gene, following protocols adapted from Olu-Le Roy et al. (2007). Whole 10–50 mg of mussel tissue was incubated in 0.5 mL of CTAB lysis buffer (2% CTAB, 1 M NaCl, 1% PVP, 20 mM EDTA pH 8, 100 mM Tris-HCl pH 8, and 0.1 mg·mL⁻¹ proteinase K) at 60°C until complete lysis. Genomic DNA was extracted using a standard phenol–chloroform protocol and precipitated with isopropanol. DNA pellets were resuspended in sterile molecular-grade water and stored at –20°C. PCR amplification was conducted using primers BathCOI-F (5′-GTGGTCTGGAATAATTGGAAC-3′) and BathCOI-R (5′-ATAAAAAGATGTATTRAARTGACG-3′). Thermocycling conditions included an initial denaturation at 94°C for 2 min; 5 cycles at 94°C for 35 s, 48°C for 35 s, and 72°C for 70 s; followed by 30 cycles at 94°C for 35 s, 52°C for 35 s, and 72°C for 70 s; with a final extension at 72°C for 10 min. PCR reactions (25 µL) included 1× PCR buffer, 2.2 mM MgCl₂, 0.5 mM each dNTP, 0.55 µM each primer, 0.02 U Taq polymerase (GoTaq, Promega), and ~20 ng of template DNA. Amplified products were purified and Sanger-sequenced using a 3730XL DNA Analyzer (Thermo Fisher Scientific) at Macrogen Europe (France). Sequences were aligned and identified using BLAST searches against GenBank.