Enhanced biodiversity of gut flora and feed efficiency in pond cultured tilapia under reduced frequency feeding strategies

Nanodrop (Thermo Fisher Scientific, Inc., Waltham, MA). The extracted rRNA was stored at -20˚C for sequencing library preparations. Prokaryotic 16S and eukaryotic 18S rRNA sequencing library preparation Prokaryotic 16S and eukaryotic 18S rRNA gene amplicons were prepared following the 16S Metagenomic Sequencing Library Preparation protocol for the Illumina MiSeq system with some modifications. Primers were designed to amplify the V3 to V5 regions of 16S rRNA [30– 31] and the V9 region of 18S rRNA [32–33] with overhang adapter sequences compatible with the Illumina index and sequencing adapters. This allowed for double indexing to increase the accuracy of the multiplexed reads (S1 Table). Amplicon PCR was used to amplify the region of interest from the gDNA extracted from the tilapia fecal material samples. A Nile tilapia-specific blocking primer (S1 Table) was introduced during the 18S amplification step to reduce the amplification of tilapia 18S rRNA and increase the amplification of lower abundance eukary- otic rRNA present in the samples. The PCR was performed as follows: 1 cycle of 95˚C for 3 min; 25 cycles each of 95˚C for 30 s, 55˚C for 30 s, and 72˚C for 30 s; 1 cycle of 72˚C for 5 min; and hold at 4˚C. Clean-up of the PCR amplicon products to remove free primers and primer dimers was performed using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA). Fresh 80% ethanol was prepared prior to clean-up. Following amplicon clean-up, index PCR was performed to attach indices to the amplicon PCR products. Dual-index primers were

designed so that samples could be multiplexed in one MiSeq lane (S1 Table). Index PCR was performed as follows: 1 cycle of 95˚C for 3 min; 8 cycles each of 95˚C for 30 s, 55˚C for 30 s, and 72˚C for 30 s; 1 cycle of 72˚C for 5 min; and hold at 4˚C. Clean-up of the PCR index prod- ucts was performed as above. All indexed amplicon concentrations were normalized and amplicons pooled into a single tube. The pooled library was verified for quality and quantified using a High Sensitivity DNA chip on the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA). The library was diluted and combined with a PhiX Control library (v3) (Illumina, San Diego, CA) at 10%. The library was sequenced on an Illumina v3 300PE MiSeq run, using standard sequencing protocols at the Genomic Sciences Laboratory at North Carolina State. Base calls were generated on-instrument during the sequencing run using the MiSeq Real Time Analysis (RTA 1.18.54) software and fastq generation. Demultiplexing, adapter trimming, and quality filtering were performed by the MiSeq Reporter Software (2.4 and 2.5.1). The library was run on two lanes to increase the number of reads for each sample. Sequence and statistical analyses The resulting demultiplexed reads were processed using the QIIME 1.9.1 toolkit [34]. Briefly, the paired end reads were joined together and UCLUST [35] was used to search against Green- genes 13_8 reference database [36] for 16S (prokaryote) analysis and SILVA release 119 refer- ence database [37–39] for 18S (eukaryote) analysis, both filtered at 97% identity. Reads not

matching a reference sequence were removed from analysis. OTUs were assigned based on a database hit of 97% or greater sequence identity and taxonomy was assigned against the appro- priate database. Core diversity analysis was used to perform α-diversity and β-diversity and their rarefaction functions. Weighted and unweighted Unifrac distances [40] were used to compute the between sample diversity which was visualized using principal coordinates analy- sis (PCoA) plots with Emperor [41]. Significant differences between treatment groups where pond was the experimental unit were determined by one-way ANOVA (Analysis of Variance) followed by Tukey’s post-hoc tests or two-way ANOVA analysis followed by pairwise comparisons using t-tests using SPSS (Statistical Package for Social Science) version-16.0, JMP 12 (SAS Institute, Cary, NC), PLOS ONE Enhanced biodiversity of gut flora and feed efficiency in pond cultured tilapia PLOS ONE | https://doi.org/10.1371/journal.pone.0236100 July 23, 2020 6 / 23