16S rRNA gene metabarcoding of Pseudosuccinea columella microbiome
DNA extraction from snails and environmental samples   All pre-PCR procedures were performed under a sterile PCR hood, decontaminated before and after use with 10% bleach, 70% ethanol, and DNA Away, followed by 20 min of UV exposure. Reusable materials were cleaned using the same protocol.  Environmental DNA (eDNA) from sediment samples was extracted using the PowerSoil Pro Kit (Qiagen) according to the manufacturers instructions. Water samples and negative controls (bottled water filtered in situ) were extracted using an adapted protocol. Briefly, filter membranes containing eDNA were cut on a sterile Petri dish with sterile tools, placed into Power Bead Pro tubes, snap-frozen in liquid nitrogen, and disrupted in a MagNA Lyser (Roche Diagnostics) for 3  30 s at 7000 rpm, with refreezing between cycles. After adding 750 L of CD1 solution, two additional agitation cycles were performed, and the samples were then processed following the kit protocol. DNA from whole snail specimens was extracted using the AllPrep Universal DNA/RNA/miRNA Kit (Qiagen). P. columella snails preserved in RNAlater were dried, then homogenized in 600 L RLT Plus Lysis Buffer with -mercaptoethanol using a TissueLyser II (Qiagen; 25 Hz, 5 min, two cycles). Tools and surfaces were changed or decontaminated between specimens using water, 70% ethanol, and DNA Away. Homogenates were snap-frozen in liquid nitrogen and stored at 80 C for up to three days. DNA was extracted following the manufacturers instructions, and concentrations were measured by absorbance at 260 nm using a BioTek Epoch reader (Agilent). Blank water samples and a ZD6310 bacterial mock community (ZymoBiomics) served as negative and positive extraction controls, respectively. PCR Conditions and 16S rRNA Gene Metabarcoding The microbiome of G0 and G2/G4 snails was analyzed by 16S rRNA gene (V3V4 regions) metabarcoding following the standard Illumina two-step protocol (32). Libraries were generated using primers 341F (5-CCTACGGGNGGCWGCAG-3) and 805R (5-GACTACHVGGGTATCTAATCC-3) with Illumina adapters. Each 30 L PCR reaction contained 15 L Q5 High-Fidelity Master Mix, 1 M of each primer, 2 L of DNA template (30 ng), and nuclease-free water. Thermocycling conditions were: 98 C for 5 min; 25 cycles of 98 C for 45 s, 60 C for 15 s, and 72 C for 10 s; followed by a final extension at 72 C for 7 min. Mock communities (ZD6310 and ZD6311; ZymoBiomics) served as positive controls for extraction and PCR, while negative controls contained nuclease-free water instead of DNA.  Of 292 samples amplified (including 12 mock communities), 257 showed clear single bands on 2% agarose gels and were retained for indexing (respectively 11 and 28 field and laboratory samples were dismissed). A second PCR (8 cycles) was performed using the Nextera XT Index Kit (Illumina) following manufacturer instructions. Indexed libraries were normalized with SequalPrep plates (Thermo Fisher Scientific), pooled, and purified using the JetSeq Clean protocol (Bioline, UK). Library quality and concentration were assessed with a Bioanalyzer High Sensitivity DNA Kit (Agilent, USA) and Qubit fluorometer (Thermo Fisher Scientific, USA). Paired-end sequencing (2  250 bp) was performed on an Illumina MiSeq platform using a MiSeq Reagent Kit v2 at the BioEnvironnement platform, University of Perpignan Via Domitia (France).