Integrating cultivation-dependent and independent methods to assess the prokaryotic diversity and secondary metabolism of the octocoral (Leptogorgia sarmentosa) holobiont under contrasting oxygen regimes

This study analyses the total prokaryotic community and the culturable bacterial fraction associated with the temperate octocoral Leptogorgia sarmentosa, collected off the coast of Sesimbra (Portugal). L. sarmentosa fragments were collected by SCUBA diving and processed (i) by direct DNA extraction (D) and (ii) by indirect DNA extraction, in which coral branches were first homogenized in calcium- and magnesium-free artificial seawater (CMFASW) using a sterile mortar and pestle, and microbial pellets (MP) were retrieved through differential centrifugation before total DNA extraction. The coral homogenates were further used for cultivation of heterotrophic bacteria in 1:2 diluted Marine Agar medium under three different oxygen regimes (aerobic (PWA), microaerophilic (PWM), and anaerobic (PWN) culturing conditions). After four weeks of incubation, all biomass on culture plates was collected using a Plate-Washing (PW) strategy, and the total DNA of the culturable bacterial fraction was extracted. All DNA extractions were performed with the Qiagen PowerSoil Pro Kit. Thereafter, high-throughput 16S rRNA gene amplicon sequencing (V4 region) was employed using Illumina MiSeq (2 x 300 bp) technology with 600 cycles.