Effects of woody forages on biodiversity and bioactivity of aerobic culturable gut bacteria of tilapia (Oreochromis niloticus)

per cage. Each forage was assigned in 4 replicate cages. The fish were fed five different diets two times daily for 8 weeks [30]. During the period, we will check the pH value, temperature, dissolved oxygen content and ammonia nitrogen content of the net cage every 6 hours and keep these values in the following range: the pH value and temperature were 7.6–7.8 and 25– 32˚C, respectively, the dissolved oxygen content was greater than 4 mg/l and the ammonia nitrogen content was less than 0.1 mg/l. Sample collection At the end of the feeding trial, feed was withheld from fish for 24 h. Additionally, three fish from each treatment group were anesthetized by tricaine methane sulphonate (MS 222, 100 ppm) to collect the gut samples [31]. Three fish from each group were randomly selected for aerobic culturable bacterial isolation. Under aseptic conditions, the gut of each fish without intestinal contents were collected in frozen tubes and stored under -80˚C. Bacterial isolation and comparison Approximately 100 mg of the thawed intestinal sample was thoroughly ground with1 ml of sterile water. After being diluted 10-fold, 0.1 ml of the bacterial fluid was spread onto prepared MRS [32] (enzymatic digest of casein 10 g/l, meat extract 10 g/l, yeast extract 4 g/l, triammo- nium citrate 2 g/l, sodium acetate 5 g/l, MgSO4�7H2O 0.2 g/l, glucose 20 g/l, MnSO4�4H2O 0.05 g/l, tween-80 1.08 g/l, dipotassium hydrogen phosphate 2 g/l), LB [33] (peptone 10 g/l, yeast extract 5 g/l, sodium chloride 5 g/l) and PIYCA (peptone 5 g/l, iron phosphate 0.1 g/l,

yeast extract 1 g/l, casein 5 g/l) agar plates (at least three plates per medium). The plates were cultured for 2 days at 30˚C to observe the bacterial morphology. According to the morphology, some isolates could be distinguished. Subsequently, different bacterial isolates were selected and transferred to new LB agar plates that were incubated at 30˚C to pure cultures [34]. Molecular characterization and identification of the bacterial isolates Take a single colony and add 5mL of DNA extraction buffer (100mmol / L Tris-HCl, 100mmol / L EDTA-Na2, 1mol / L NaCl, 1% CTAB pH 8.0), then add 10% SDS 800μL and mix and incubate at 37˚C for 1h. During this period, the mixture was continuously mixed several times, and an equal volume of saturated phenol: chloroform: isoamyl alcohol (25: 24: 1) was added to the centrifuge tube and centrifuged at 12 000 r / min for 10 minutes and extracted twice, and then chloroform: isoamyl alcohol (24: 1) Centrifuge at 12 000r / min for 10min and extract once. Take the supernatant in a new centrifuge tube and add 0.6 volumes of isopropanol to precipitate DNA for 1h, then centrifuge at 12 000r / min for 10min and discard the superna- tant. The DNA pellet was dissolved in 0.1 mL TE buffer (10 mmol / L Tris-HCl, 1 mmol / L EDTA, pH 8.0 with RNase enzyme), and then heated at 100˚C for 30 min [35, 36]. The mixture was then centrifuged at 10,000 r/m for 10 min at 4˚C to obtain the template DNA. The bacterial universal primers 27F (50-AGAGTTTGATCCTGGCTCAG-30) and 1492R (50-GGTTACCTTG

TTACGACTT-30) were used to PCR amplify the 16S rDNA sequence (Eppendorf AG 22331 Hamburg 6331). PCR amplifications were performed with an initial denaturation temperature of 95˚C for 5 min; followed by 30 cycles of 95˚C for 60 s, 55˚C for 60 s (primer annealing) and 72˚C for 90 s (primer extension), with a final primer extension step at 72˚C for 10 min [37]. Sequencing of the 16S rDNA of the selected isolates was performed by BIOTREE (China). Sequences that were obtained using National Center for Biotechnology Information (NCBI) databases were compared with Basic Local Alignment Search Tool (BLAST) searches [38]. When the relatively best matches were from the same species and were 95% similar to the query sequence, this species name was assigned to the selected isolate [39]. PLOS ONE Effects of wood forages on gut bacterial biodiversity and bioactivity in tilapia PLOS ONE | https://doi.org/10.1371/journal.pone.0235560 July 2, 2020 4 / 17