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

consumed in 2014 [2]. Currently, tilapia and many other fish species are grown predominantly in pond culture under various conditions: intensive culture with high stocking densities, reli- ance on commercial feeds, and often requiring mechanical aeration of the ponds; extensive cul- ture where fish are stocked at low densities and organic and inorganic fertilizers stimulate pond primary food production with little to no additional feed supplementation; and, most fre- quently, semi-intensive culture conditions in which fish are stocked at moderate densities and provided supplemental feeds to complement microorganisms produced naturally with pond fertilization, which can effectively quadruple production over extensive culture systems [3–5]. Feed is the major expense in aquaculture, comprising up to 50–70% of total production costs for tilapia. Methods to limit the amount of feed can help reduce overall costs to producers and has major impacts on small-scale farmers that dominate in under-developed countries. Reductions in feed ration by up to 50% by using alternate-day feeding improves the feed effi- ciency and economic returns of tilapia monocultured at densities of 2–4 fish/m2 with little impact on growth, survival, or production yield [6]. A better understanding of how finfish acquire and utilize nutrient inputs is requisite for future improvements in aquaculture production efficiency. The underlying mechanism explaining how reduced frequency or alternate-day feeding strategies can achieve equivalent

production yields with less feed is poorly understood in finfish. Some evidence suggests that during periods of fasting, nutrient uptake efficiency in the intestine is intrinsically enhanced, leading to a more-efficient uptake of nutrients at the next feeding period [7–10]. Thus, fish being fed a daily regime have lower uptake efficiency and do not receive maximal dietary bene- fit. The increase in nutrient uptake efficiency following periods of decreased nutrient availabil- ity has been postulated, in part, to explain the compensatory growth (CG) response observed in many aquaculture species [7–11]. Additionally, reduced feeding may promote foraging on pri- mary food production within the ponds, leading to a more diverse diet (e.g. algae, insect larvae, plankton), enhancing nutrient recycling within the ponds. This enhanced diversity may directly influence intestinal absorption by promoting colonization and growth of microbes that may aid in nutrient utilization and uptake efficiency that are key to growth and health of the fish [12]. The emerging field of metagenomics has substantial implications for sustainable aquacul- ture, as diet, feeding strategy, and other environmental factors strongly influence the diversity and constitutive abundance of intestinal microbiota in both humans and fish [12–15]. In fin- fish, research has shown that probiotic maintenance of beneficial gut flora can promote growth, greater nutrient availability, and better stock health [16–17]. Early studies in channel

catfish (Ictalurus punctatus) and carp (Cyprinidae) identified several nutrients (e.g. biotin, pantothenic acid, vitamin B12), which are produced by intestinal microbes, but may be limit- ing in lesser-quality feeds [18–19]. Interestingly, proper intestinal flora in tilapia may also posi- tively impact human health as natural flora inoculates could theoretically out-compete non- natural pathogenic microbes. In Nile tilapia cultured in fertilized ponds found to be contami- nated with fecal coliform bacteria in Bangladesh, E. coli comprised up to 10% of fecal coliform bacteria identified by classic plating techniques, which could be passed on to consumers through improper storage and handling practices [20]. Absorption of nutrients from feed is facilitated through membrane bound transporters located within enterocytes of the gastrointestinal lumen [21–22]. Nutrient transporters are sol- ute-linked nutrient carriers with roles in transporting and exchanging nutrients, such as amino acids and sugars, and ions across epithelial cellular membranes [23]. Upregulation of nutrient transporter gene expression has been associated with enhanced utilization efficiencies of nutrients from the gut [24] and could play an important role in increasing uptake in tilapia fed at reduced frequencies [6, 25]. Deficiencies in key nutrients may also lead to upregulation of nutrient transporter gene expression or activity resulting in a greater ability to utilize 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 2 / 23 Accession No. PRJNA485292. All other relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the AquaFish Innovation Lab through the United States Agency for International Development/Oregon State University (USAID EPP-A-00-06-00012-00) and by participating institutions, particularly North Carolina State University and Bangladesh Agricultural University and the NSF (IOS-1457040 to RJB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.