16S rRNA gene amplicon sequencing of gut digesta of marine fish fed a CHT5 diet, replicate 2 [JMF-2002-2-0008A]
To access gut microbiota modulation due to dietary IM, five isoproteic (45%) and isolipidic (18%) diets were used: a control diet (CTR); a diet similar to the CTR with 5% commercial chitin (CHIT5), and three diets with 25% inclusion of HM25, TM25 and HEM25, respectively. The experimental diets were assigned to 3 groups of 15 fish with an initial mean body weight of 53.7 +/- 2.67 g. The trial was conducted in a recirculating aquaculture system equipped with 15 fiberglass tanks of 100 L water capacity, thermo-regulated to 22.7 +/- 0.3C and supplied with a continuous flow of seawater (36.0 +/- 0.5 g L-1 salinity, circa 7 mg L-1 oxygen). The photoperiod was set, with artificial illumination, to 12:12h light:dark. The trial lasted 8 weeks, and sh were fed by hand twice a day, 6 days per week, until apparent visual satiation. Two fish per tank were randomly sacrificed with an anesthetic overdose (3.0 mL L-1 ethylene glycol monophenyl ether), 4h after the first meal. Whole-gut (without pyloric caeca) was aseptically excised and squeezed to collect the digesta contents. Mucosa samples were obtained by scraping the internal intestinal mucosa after opening the intestines in their longitudinal axis. To overcome inter-fish variation, the resulting material from the two fish was pooled into one sample per tank, immediately frozen in liquid nitrogen and stored at -80C until further analysis to assess differences between dietary groups. DNA was extracted from 250 mg of either the collected gut contents or gut mucosa, which were transferred to 2 mL sterile tubes containing 400 mg of 425-600 um glass beads (Sigma-Aldrich, Germany) and 500 uL of STE buffer. The samples were homogenized in a Precellys homogenizer (Bertin Instruments, France) using 3 cycles of 60 s at 4500 rpm with 60 s intervals on ice between cycles. The samples were incubated for 15 min at 75C, centrifuged (16000 g for 1 min at 4C) and 500 uL of supernatant was collected to a new 2mL sterile tube. DNA was extracted from the supernatant as described before (https://pubmed.ncbi.nlm.nih.gov/33544251/). Nucleic acid pellets were finally resuspended in 50 uL of nuclease free water. As a negative control for the extraction procedure, a sample with only lysis buffer was processed in parallel with all samples. DNA were subjected to a 2-step PCR barcoding of the V4 region of the 16S rRNA gene (https://pubmed.ncbi.nlm.nih.gov/34093488/).