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

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

S1 Table. Primers used in studies for Nile tilapia (O. niloticus) subjected to reduced-feed- ing regimens. (XLSX) S2 Table. Water quality parameters of ponds used in studies for Nile tilapia (O. niloticus) subjected to reduced-feeding regimens. Values are mean ± SD. Values with different letters are significantly different (P < 0.05). (XLSX) S3 Table. Plankton abundance in ponds used in studies for Nile tilapia (O. niloticus) sub- jected to reduced-feeding regimens. Values are mean ± SEM. Values with different letters are significantly different (P < 0.05). (XLSX) S4 Table. Proportions by treatment of prokaryotes and eukaryotes identified to the family level in fecal material from Nile tilapia subjected to reduced-feeding regimens. (XLSX) S5 Table. Unique prokaryotic Operational Taxonomic Units (OTUs) identified in the fecal material from Nile tilapia subjected to reduced-feeding regimens. (XLSX) Acknowledgments The authors would like to thank Dr. Hillary Egna of Oregon State University for her leadership of the Aquafish Innovation Lab and her intellectual contributions to the work. The opinions expressed herein are those of the authors and do not necessarily reflect those of the AquaFish Innovation Lab-USAID or the NSF. Author Contributions Conceptualization: Md. Abdul Wahab, David A. Baltzegar, Russell J. Borski. Data curation: Scott A. Salger. Formal analysis: Scott A. Salger. Funding acquisition: Md. Abdul Wahab, Russell J. Borski. Investigation: Jimi Reza, Md. Abdul Wahab, Alexander T. Murr, Russell J. Borski.

Methodology: Scott A. Salger, David A. Baltzegar, Alexander T. Murr. Project administration: Md. Abdul Wahab, Russell J. Borski. Resources: Md. Abdul Wahab, Russell J. Borski. Supervision: Md. Abdul Wahab, Russell J. Borski. Validation: Scott A. Salger. Visualization: Scott A. Salger. Writing – original draft: Scott A. Salger. Writing – review & editing: Scott A. Salger, Courtney A. Deck, Russell J. Borski. 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 19 / 23

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