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

establishment of beneficial gut flora to increase nutrient absorption is an emerging research focus in human biology and aquaculture science [17] and may serve to augment existing prac- tices of sustainable feeding and reduction in environmental footprint. The most abundant phylum of prokaryotes identified in all treatments were the Firmicutes. The Firmicutes are a group of mostly Gram-positive bacteria and are important members of the gut microbiome of many vertebrates. The proportions of Firmicutes identified declined with reduced application rates of commercial feed in fertilized ponds (Fig 3). An increase in the relative abundance of Firmicutes is indicative of obesity in mammals and positively corre- lated with caloric intake in bony fishes [64–66]. The higher proportion of Firmicute bacteria in tilapia from treatments fed daily in fertilized ponds in our study indicates that there may be an abundance of high-calorie food sources available to these fish. Bacteria from the classes Fuso- bacteriaceae and Clostridiaceae were most abundant in the fecal material of Nile tilapia used in our study. These groups include many pathogenic strains that could cause disease with immu- nosuppression or injury to the gut epithelium [67–68]. Cetobacterium somerae (a Fusobacter- iaceae) had the highest number of reads and proportional abundance in our study with no apparent trend in regulation by feeding or fertilization. C. somerae is an indigenous bacterium in the intestinal tract of cultured freshwater fish and is highly efficient at producing Vitamin

B12, an essential micronutrient [69–70]. The highest growth and survival rates were observed in T1 (daily feeding along with pond fertilization), T2 (alternate day feeding along with pond fertilization), and T5 (daily feeding only). We identified 7 OTUs (corresponding taxonomic assignments: Acutodesmus sp., Cyano- bacterium sp., Bacillus sp., Blautia sp., Anaerovorax sp., Sphingomonas sp., and Desulfococcus sp.) that were common between all three of these treatments. The identification of the species relating to these OTUs are currently unknown. Future work will be necessary to determine not only the species but also the exact functions of these microorganisms and to determine whether they might serve to benefit growth of Nile tilapia. Six families of eukaryotic phytoplankton and four groups of zooplankton were identified from the ponds in this study. Of those identified, plankton from the Families Bacillariophy- ceae, Chlorophyceae, Euglenophyceae, Rhodophyceae, Subphylum Crustacea, and Phylum Rotifera were also identified in the microbial communities in the fecal material from the Nile tilapia in these studies. Although the microscopic analysis used for pond water only allowed us to identify plankton that were large enough to be viewed, the commonalities with the fecal matter indicate that the tilapia were consuming plankton from the pond as an additional food source. The Holozoans were the dominant group of eukaryotes identified in Treatments 1 (daily feeding along with pond fertilization), 3 (every third day feeding along with pond fertili-

zation), 4 (pond fertilization only), and 5 (daily feeding only) with the most abundant organ- isms within this group being the Rotifers. As part of the zooplankton community in the ponds, the rotifers make up a large proportion of the food available to the tilapia, outside of commer- cial feeds. By contrast, T2 (alternate day feeding along with pond fertilization) was dominated by the group Chloroplastida which includes green plants and algae. The Chloroplatids made up a large proportion of eukaryotes identified in all treatments and the most highly identified sequences within this group aligned to Zea mays. Zea mays could be a component of the feed used in these trials but OTUs were also found in large proportions in the guts of the fish from T4 (pond fertilization only) which did not receive any commercial feed. Cholophytes were also identified as part of the phytoplankton community using microscopic analysis. As such, these OTUs may align to other plant sequences similar to Zea mays, that are not found in the avail- able database. The other groups with highest proportions identified across all treatments were the groups Alveolata (ciliated protozoans and dinoflagellates) and Stramenopiles (diatoms and algae). All of these would be components of the natural production in established ponds 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 17 / 23