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

fertilization (T1) and lowest in the fertilization alone group (T4). Both nitrites and nitrates were higher in fertilized ponds (T1-T4) and lowest in non-fertilized, fed daily ponds (T5). Dis- solved oxygen levels were highest in ponds either fed every 3 days with fertilization or those fertilized only (T3 and T4) and lowest in ponds fed daily independent of whether ponds were fertilized or not (T1 and 5). The pH was highest in the ponds that were fertilized only (T4) and lowest in ponds that were fed a commercial diet (T1-T3, T5). Six Families of eukaryotic phytoplankton and four groups of zooplankton were identified over the course of the pond trials (S3 Table). The eukaryotic phytoplankton Families identified were Bacillariophyceae, Chlorophyceae, Cyanophyceae, Euglenophyceae, Rhodophyceae, and Xanthophyceae and zooplankton were Copepoda, Crustacea, Rotifera, and Cladocera. T5 had significantly higher counts of the phytoplankton Family Euglenophyceae (4035.7 ± 466.8 SEM cells/L) whereas T1 had the lowest counts of this family (1750.0 ± 252.5 SEM cells/L; P = 0.0215). There was no difference in total eukaryotic phytoplankton between treatments (P = 0.0851). For zooplankton, T1 had significantly higher counts of Phylum Rotifera (6482.1 ± 1291.0 SEM cells/L; P < 0.001) and Order Cladocera (1464.3 ± 198.4 SEM cells/L; P = 0.0001) and significantly higher total zooplankton (11571.4 ± 1854.9 SEM cells/L; P = 0.0003) than the other treatments. There was no difference in total plankton between any of the treatments (P = 0.221).

Effect of feeding frequency on intestinal nutrient transporter expression The mRNA levels of six intestinal nutrient transporters were measured via real-time qPCR in the anterior intestine of fish from the growth trial. The nutrient transporters evaluated were: (1) facilitated glucose/fructose transporter (slc2a5), (2) facilitated 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). The efficiency of all real-time qPCR reactions were between 90–110% and the stan- dard curve correlations were greater than 0.97 for all assays performed. Our resulting gene expression values (copy number / ng Total RNA) were normalized to 18S rRNA levels (no sig- nificant difference between treatments was observed in the 18S rRNA gene). There was no significant difference in gene expression of the 6 nutrient transporters ana- lyzed between treatments where supplemental food and pond fertilization was provided (T1—T3) regardless of the frequency of feeding, except that slc38a2 was significantly lower in T2 than T1 and T3 fish (Fig 2). Gene expression for the slc2a5, slc2a6, slc27a4, and slc38a2 transporters was significantly higher in fish from T4 (pond fertilization only) than T5 groups (daily feeding only). There was no difference among treatments in the mRNA abundance of the slc38a4 and slc43a1 transporters (Fig 2e and 2f). Effect of feeding frequency on the gut microbial community—prokaryotic assessment

Following quality filtering, a total of 715,725 paired end sequences (330,883 for 16S; 384,842 paired end sequences for 18S) were obtained from the V3—V5 regions of 16S rRNA (prokary- ote) and the V9 region of 18S rRNA (eukaryote). All resulting Fastq files can be accessed in the National Center for Biotechnology information (NCBI) under GenBank database BioProject Accession No. PRJNA485292. A total of 20 prokaryotic phyla, 43 classes, 92 orders, and 145 families were found in the fecal material of tilapia (100% bacteria, no archaea). The dominant phyla identified from these samples belonged to the Fusobacteria (80.4%), Firmicutes (13.8%), Cyanobacteria (2.4%), Bac- teroidetes (1.3%), and Proteobacteria (1.3%) (Fig 3a, S4 Table). The proportions of the 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 9 / 23