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

catfish, readily feed on diverse food sources throughout their life, including phytoplankton and zooplankton or other natural pond foods enhanced through fertilization or application of supplemental formulated feeds. Hence, tilapia fed on alternate or even every third day likely forage at a greater rate between meals on pond organisms produced through enhanced nitro- gen and phosphorous inputs from fertilizers or feeds than fish fed daily. Less reliance on sup- plemental feeds will increase overall feed efficiency as demonstrated here with low FCR, albeit at a detriment to growth when animals are fed at a frequency of every three days. Clearly, suffi- cient nutrients, derived in this case from formulated feed, is essential to maximally stimulate growth as we found alternate day fish achieved weights and lengths similar to daily fed fish while those fed every third day did not. The responses to alternate day feeding we observed here with studies in Bangladesh corroborate those previously shown in the Philippines on commercial farms where tilapia were stocked at a 20% lower density and fed a formulated diet composed of high levels of fishmeal and rice bran [6]. Water quality parameters are affected by excess nutrients, but small variations within normal range are unlikely to account for any of the changes in tilapia growth observed in this study. All water quality parameters were well within the suggested range for growing tilapia in ponds (S2 Table) [4, 44]. Collectively, the findings indicate that addition of supplemental feeds can enhance growth of tilapia by 2–4 fold

depending on the frequency of feed application and that alternate day feeding is the most cost- effective feed management method for maximizing growth and yield of tilapia in semi-inten- sive culture, a strategy that is likely to be applicable for pond production of tilapia throughout the world. Reduced feeding strategies can achieve equivalent production yields with less feed; however, the mechanisms are poorly understood [7–11]. Absorption of nutrients from feed is facilitated through membrane bound transporters located within enterocytes of the gastrointestinal lumen [21–22]. We evaluated if changes in expression of key solute-linked nutrient carriers (SLC), whose abundance may impact nutrient absorption efficiency [21] might be associated with the changes in feed efficiency and growth of Nile tilapia shown here under the different feeding protocols. We used the proximal (anterior) portion of the small intestine to measure solute transporter gene expression levels. Tilapia have a long, coiled intestine in order to pro- cess and absorb nutrients from their mainly herbivorous or omnivorous diet. Tilapia lack a pyloric caecum, thus the small intestine is the region of highest nutrient absorption, especially in the proximal area which is the most developed region of the small intestine and exhibits the highest enzymatic activity and absorption of nutrients [46–47]. A number of ATP-binding cas- sette (ABC) efflux transporter genes associated with multi-drug resistance and efflux of xeno-

biotics are also expressed in the proximal small intestine of Nile tilapia [48] and SLCs are expressed in a similar region of the intestine in zebrafish (Danio rerio) [49]. The expression of solute transporters slc2a5, slc2a6, slc27a4, and slc38a2 in the proximal intestine of the Nile tilapia were significantly higher in fish grown in ponds where only fertiliz- ers were applied (T4) than in fish grown in ponds which were fed daily without the addition of fertilizers (T5) (Fig 2). The seemingly low expression of all transporters examined in the intes- tines of fish fed daily without pond fertilization (T5) may in part be the result of receiving simi- lar quantities of feed with the same nutrient profile day in and day out, hence preventing the need for any adaptive response from the intestine [50]. It may also be due to higher rates of intestinal growth in these fish. Increased food consumption can induce lengthening of the intestine and proliferation of the villi to enhance nutrient uptake as well as increase cell turn- over rates. This in turn can increase the proportion of immature, non-transporting cells and cause a non-specific change in overall transporter abundance [50]. Conversely, the relatively high expression of these transporters in fish grown in ponds where only fertilizers were applied (T4) likely indicates that gaining nutrition from natural flora and fauna boosted by fertilization 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 15 / 23