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

either fertilization or through the process of breakdown of feed and fish waste. Thus, it is apparent that the tilapia in this study were feeding on a wide variety of eukaryotic organisms in the ponds. Being omnivores, tilapia will utilize any food resources available to them for growth and as such a decrease in the application frequency of the commercial feed would likely increase the rates of consumption of natural food sources, whether that be planktonic sources or plant material growing in the ponds. As seen here (Fig 5), tilapia in T2 (alternate day feeding along with pond fertilization) had the greatest diversity of eukaryotic organisms identified in their guts. This could have led to a greater variety of nutrients available to them for growth than the tilapia in the other treatment groups. Alpha diversity measures are indices of the diversity within a community. The Chao 1 index is commonly used to estimate the total number of species within a community and is based on the number of rare OTUs in that community [71]. The diversity of prokaryotes was significantly higher in Treatments 1 (fed daily with pond fertilization) and 2 (alternate day feeding with pond fertilization) than in the other three treatments while T2 had the highest diversity of eukaryotes and Treatments 3 (fed every third day with pond fertilization) and 5 (daily feeding only) had the lowest. It is predicted that ecosystems with high species-richness better utilize limiting resources [72–73] and are more resilient to disease states such as inflam-

matory bowel disease [74] and Clostridium difficile-associated pathologies [75]. Thus, increased diversity in the gut microbiome could enhance health, survival, and feed efficiency in farmed tilapia. As T5 (daily feeding only) also received daily feeding but had significantly lower Chao 1 indices relative to T1 (fed daily with pond fertilization), it is likely that pond fer- tilization alone is beneficial for growing tilapia as it increases the availability of natural food sources providing a more varied diet. Alternate day feeding (T2) appears to also be beneficial for enhancing microbe diversity as these fish are likely consuming a greater proportion of plankton and plant materials than those fed daily (T1). Conclusion It is estimated that 50–70% of total variable costs for growing tilapia is attributable to formu- lated feeds. This study demonstrated that alternate-day feeding reduces the costs of feeds for growout of tilapia by 50%, increases feed efficiency by almost 76%, and has little impact on growth, survival or yield of tilapia farmed in ponds. The results also suggest that tilapia grown in fertilized ponds without supplemental feeds may be nutritionally impaired as key nutrient transporters in the gut are enhanced in preparation for increased uptake of solutes should food become available, a process that is mitigated when animals are provided supplemental feeds. The intermediate expression of gut nutrient transporters in alternate-day fed tilapia may reflect a condition for the most efficient uptake of nutrients from the GI tract. Finally, this

work shows that the use of fertilizers and the implementation of an alternate-day feeding strat- egy increase the diversity of intestinal microbiota that may function in promoting nutrient assimilation and in these fish. These microbes may serve as promising candidates for isolation and development of probiotics beneficial to increased feed efficiency, growth, and health in tilapia. Supporting information S1 Fig. Chao1 rarefaction plots of microbial communities identified from the anterior intestine of Nile tilapia subjected to reduced-feeding regimes (mean ± SEM). (a) 16S rRNA (prokaryotes); (b) 18S rRNA (eurkaryotes). (TIF) 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 18 / 23