Characterisation of the microbiome of Halichondria panicea in the Baltic Sea using 16S rRNA gene amplicon sequencing

In this study, we sequenced the microbiome of 40 Halichondria panicea individuals from four locations in the Baltic Sea. The main goal was to investigate how the prokaryotic community inside the sponges responds to changes in salinity across the salinity gradient of the Baltic Sea. Sponge tissues and seawater were sampled on 2018-05-18 in Sweden (1 location) and between 2023-04-04 and 2023-04-23 in German waters (3 locations). At each site, 10 individuals were sampled by cutting off a thumb-sized piece of sponge tissue. Three seawater samples were taken per location in close proximity to the sponge colonies, and temperature and salinity were recorded. The sponge tissue was cleaned of visible contaminants such as ingrown algae or mussels and rinsed in sterile seawater before DNA extraction using the DNeasy PowerSoil Pro Kit (Qiagen). Input for DNA extraction was 100-200 mg of wet tissue. For the seawater samples, 500 mL of each sample collected on 0.22 µm pore-size polycarbonate filters, were extracted using the DNeasy Mini Kit (Qiagen). The V3-V4 hypervariable region of the 16S rRNA gene was PCR-amplified using primers 341F (3'-CCTAYGGGRBGCASCAG-5') and 806R (3'-GGACTACNNGGGTATCTAAT-5') (Sundberg et al., 2013). The copy numbers of the 16S rRNA gene for Ca. Halichondribacter symbioticus and for the total bacterial community were determined by droplet digital PCR (ddPCR), using the primers Hal Sym F (5'-CGCGGATGGTAGAGATACCG-3') and Hal Sym R (5'-TGTCCCCAACTGAATGCTGG-3') (Schmittmann & Pita, 2022), and the universal bacterial primers E1052f (5'-TGCATGGYTGTCGTCAGCTCG-3') and E1193r (5'- CGTCRTCCCCRCCTTCC-3'), respectively. The ddPCR was performed on a QX200 Droplet Digital System by Biorad (USA) using the EvaGreen Assay.