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

Scientific, Waltham, MA, USA) at room temperature for shipment to NCSU and subsequently stored at -80˚C until total RNA extraction. All five samples per pond were pooled together prior to total RNA extraction (See Nutrient Transporter Gene Expression, below; n = 4 pooled samples per treatment; N = 20). Tilapia fecal material was collected from the posterior intestine (colon; approximately 5 cm section 1 cm anterior to anus) and rRNA was extracted using a Xpedition Soil/Fecal DNA Miniprep kit (Zymo Research Corp., Irvine, CA, USA) following the manufacturer’s protocol (see Ribosomal RNA extraction, below). Samples from 4 fish per pond were pooled together from all 4 replicate ponds for all treatment groups (n = 4 pooled samples per treatment; N = 20). This pooled sample design was used to offset potential vari- ability of microbiota within individuals, instead focusing on common patterns, which may be more reflective of changes with treatment group among the population (pond) as a whole. Water quality Water temperature (˚C), transparency (cm), pH and dissolved oxygen (mg/L) were measured weekly and total alkalinity (mg/L), ammonia-nitrogen (mg/L), nitrate-nitrogen (mg/L), nitrite-nitrogen (mg/L), phosphate-phosphorus (mg/L) and chlorophyll-a (μg/L) were mea- sured biweekly between 0900–1000 h according to previously established protocols [27]. Collection and quantification of plankton samples Ten liters of water were collected biweekly from five locations in each pond for plankton sam- pling. The collected water was filtered through a 45 μm plankton mesh screen. The filtered

sample was brought to a standard volume of 50 mL using distilled water. The plankton samples were fixed in 5% neutral buffered formalin in 100 mL bottles for subsequent studies. Plankton was quantified using a 1 mL fixed plankton sample in a Sedgewick-Rafter (S-R) cell counting chamber on a Swift M4000 binocular microscope with phase contrast capabilities. Plankton identifications were made at the Family level. Growth and production parameters Growth and production parameters were calculated as below: Weight gainðgÞ ¼ ðMean final weight of fishðgÞÞ ðMean initial weight of fish ðgÞÞ Specific Growth RateðSGR; % body weight=dayÞ ¼ Lnðfinal weightÞ Lnðinitial weightÞ Culture period in days � � � 100 Survival rate % ð Þ ¼ No:of fish harvested No: of fish stocked � � � 100 Feed Conversion RatioðFCRÞ ¼ Total feed given ðgÞ Total weight gainðgÞ Gross production ¼ ðNo:of fish harvestedÞ � ðFinal weight of fish ðgÞÞ Net production ¼ ðNo: of fish harvestedÞ � ðWeight gain of fish ðgÞÞ 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 4 / 23