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

application alone may lack sufficient nutrients necessary to maintain growth and health that would otherwise be available with commercial diets. Thus, gene expression for these transport- ers may be increased due to the lack of nutrients and for preparing the intestinal cells to rapidly take them up once they become available [50]. Interestingly, fish grown in ponds that received feed and fertilization (T1, T2, and T3), particularly those fed every two (T2) or three days (T3), had intermediate mRNA abundances of certain solute transporters (Fig 2). This suggests that they could be more efficient at nutrient uptake and utilization, particularly when commercial feeds are only periodically available, and that they may perhaps have a greater variety of trans- porters expressed at lower quantities due to an increased diversity and abundance of natural food sources in the diet. It is interesting that the fish in T1 (daily feeding with fertilization) had approximately 3-fold higher levels of these transporters than T5 (daily feeding only) as both groups were fed daily, but is likely the result of a more varied diet and greater overall abun- dance of food sources. The solute transporters slc2a5, slc38a2, and slc27a4 exhibited similar expression profiles across the five treatments. When a nutrient yields high metabolizable energy, high concentra- tions will typically stimulate an upregulation of its transporter as is the case for sugars and fatty acids. By contrast, when a nutrient can be toxic at high concentrations, it will normally cause a

reduction in the expression of its transporter [50]. GLUT5, the protein produced from the slc2a5 gene, is a facilitated fructose transporter in vertebrates [23, 51]. Although a member of the facilitated glucose transporter superfamily (SLC2), glucose appears to be a minor compo- nent of its mechanisms. GLUT5 is essential for the transport of fructose across the luminal membrane of the small intestine and transcription of slc2a5 in the intestine is rapidly increased when a high-fructose diet is consumed [52–54]. Thus, the relatively higher levels of this trans- porter in fish from ponds with fertilization only (T4) may indicate a greater abundance of fruc- tose in the natural flora and fauna relative to the commercial diet. Similarly, slc27a4 gene expression was highest in fish grown in fertilized ponds without commercial feed as well as in fish fed at different frequencies along with pond fertilization. These increases may be dictated primarily by the nutrients available in the natural products produced by pond fertilization. Slc27a4 encodes the FATP4 fatty acid transporter. It promotes lipid absorption in the mouse ileum [55] and the gene is located in a growth-related quantitative trait loci (QTL) in the orange-spotted grouper (Epinephelus coioides) [56]. The bacterium Clostridium ramosum increases slc27a4 gene expression with high-fat diets in mice [55]. Although C. ramosum was not directly identified in the guts of the Nile tilapia in this study, several sequences did align to members of Clostridium sp. OTUs.

Contrary to the fructose and fatty acid transporters, gene expression of slc38a2 increases in response to the absence of amino acids. This increase leads to the translation of the gene to make the SNAT2 protein which has been shown to aid the recovery of cell volume following amino acid starvation or hypertonic stress [57–61]. In addition, amino acid transporters may serve as sensors of nutrient supplies to regulate expression of the transporters and thus nutri- ent uptake by the cells [62]. The results show that expression of this gene is upregulated in T4 with only pond fertilization and lower in fed treatments regardless of fertilization. This implies that natural biota increased through fertilization of ponds may lack the full complement of amino acids necessary for the intestine to maintain cellular osmolality. In this case again the slc38a2 would be upregulated to improve the efficiency of absorbing amino acids under limit- ing conditions whereas in fish receiving commercial feeds that typically have adequate protein or amino acids, the transporter is expressed at lower levels to prevent excessive amino acid uptake which can lead to toxicity [50, 63]. This investigation also assessed how the gut microbial flora is altered by feeding/fertilization strategies that could potentially identify microbes beneficial to tilapia growth and health. 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 16 / 23