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

Benefit to Cost RatioðBCRÞ ¼ Total revenue Total cost Nutrient transporter gene expression From the publicly available tilapia genome assembly [28], we identified 6 candidate transport- ers putatively involved in the digestive transport of amino acids, dietary sugars, and lipids across the intestinal epithelium: (1) facilitated glucose/fructose transporter (slc2a5), (2) facili- tated glucose transporter (slc2a6), (3) long-chain fatty acid transporter (slc27a4), (4) Na+- amino acid transporter 2 (slc38a2), (5) Na+-amino acid transporter 4 (slc38a4), and (6) large neutral amino acid transporter subunit 3 (slc43a1). Tilapia gut transporter mRNA expression was quantified in anterior intestinal tissues (see Fish and Pond Sampling, above, for sampling details) using real-time quantitative PCR (qPCR) performed as previously described with a few modifications [29]. Briefly, total RNA was isolated from the anterior intestine using Tri Reagent (Invitrogen, Carlsbad, CA). One microgram of the total RNA was used to synthesize cDNA using a High Capacity cDNA Syn- thesis kit (Applied Biosystems, Carlsbad, CA) following treatment with Turbo DNA-free (Ambion, Foster City, CA). RNA was quantified and checked for quality at each step using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE) and agarose gel electrophoresis, respectively. Gene expression (mRNA) of the different transporter forms was measured in the tilapia cDNA using SYBR Green chemistry. Transporter gene-specific primers

(S1 Table; Integrated DNA Technologies, Inc., Coralville, IA) were designed using the IDT PrimerQuest Tool (Integrated DNA Technologies, Inc.). Optimization for appropriate anneal- ing temperature, primer concentrations, and cycling parameters was performed using pooled cDNA from the above reverse transcription reactions. One hundred ng of starting total RNA was used for qPCR analysis with Brilliant II QPCR Master Mix (Agilent Technologies, Inc., Clara, CA) containing 1.5 μM gene-specific primers. No template controls and no reverse tran- scription controls were incorporated into the assay. Pooled cDNA was used to produce the cDNA for creating the standard curves. All qPCR assays were run in triplicate wells on a CFX384 Real-Time PCR System (BioRad Laboratories, Inc., Hercules, CA). Cycling conditions were: 1 cycle, 50˚C for 2 min; One cycle, 95˚C for 10 min; 40 cycles, 95˚C for 15 s, 60˚C for 1 min. Melting curve analysis was performed to determine primer specificity. Data was normal- ized to the starting total RNA concentration. Gene copy number was calculated by comparing the mean cycle threshold (Ct) to the serially diluted cDNA standard curve (R2 = 0.98). The PCR efficiencies were between 90% and 110% for all reactions. The gene expression data were then normalized to the expression of 18S ribosomal RNA, whose levels were found to be simi- lar across treatment groups. Ribosomal RNA extraction Extraction of ribosomal RNA from tilapia fecal samples (see Fish and Pond Sampling, above,

for sampling details) was performed using an Xpedition™ Soil/Fecal DNA MiniPrep Kit (Zymo Research, Corp., Irvine, CA) following the included protocol. Up to 0.25 g of fecal sample was placed into a ZR BashingBead Lysis Tube with 750 μL Xpedition Lysis/Stabilization Solution. The tube was secured in an Xpedition Sample Processor and processed for 30 s and stored at room temperature until extraction. The concentration and quality were determined by 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 5 / 23