Effects of woody forages on biodiversity and bioactivity of aerobic culturable gut bacteria of tilapia (Oreochromis niloticus)

Further identification of the five candidate new species is necessary, including assays such as multigene analysis and morphological studies. Interestingly, Mikalsen and Colquhoun [57] isolated a Francisella asiatica sp. nov. from farmed tilapia from Costa Rica. These results sug- gested that the intestinal bacteria of tilapia may contain many novel bacterial species. With the rapid development of molecular biology techniques, high-throughput sequencing methods can aid in the identification of more novel microbial species from the guts of tilapia [58]. To some extent, the gut bacterial composition of tilapia from the five groups can reflect the effect of woody forage on the aerobic culturable bacterial community. For examples, the num- ber of bacterial species and isolates recovered in the MOL and fermented MOL groups was higher than that observed in the control group. This result might have occurred because of rel- atively high contents of polysaccharides and trace element [59], which could be available for more bacteria to utilize. In contrast, the number and abundance of bacterial in the FM and fer- mented FM groups was lower than that observed in the control group. Furthermore, Bacillus, a common probiotic [60], accounted for the majority of the aerobic culturable bacterial com- munity in the five groups, whereas other genera, including Fictibacillus, Lysinibacillus, Micro- bacterium, Rhodococcus, Rummeliibacillus and Thalassospira, were not isolated from the control group (Table 3). An interesting phenomenon was that after feeding with woody forage,

the abundance and quantity of Bacillus in the control group were lower than those in the woody feed group. According to Table 5, Bacillus altitudinis, Bacillus cibi, Bacillus stratospheri- cus, and Bacillus subtilis were antagonistic to selected fish pathogens, but these four Bacillus species were not found in the control group. Whether woody forage affects all this requires in- depth research. These results indicated that the woody forage enriched the diversity of aerobic culturable bacteria in the guts of tilapia. In addition, conditional pathogens such as Proteus and Stenotrophomonas were rarely observed in the guts of tilapia in the FM and fermented FM groups in this study (Table 3). These results may be associated with the antibacterial activ- ity of FM [61]. The presence of some specific bacterial species can also reflect the influences of woody for- ages on the diversity of aerobic culturable bacteria in the intestines. Some specific bacterial spe- cies were found in the guts of tilapia in this study. For example, Fictibacillus rigui, Lysinibacillus sphaericusare and Rummeliibacillu spycnus were only detected in the MOL group (Table 3). Fictibacillus rigui was first isolated from fresh water of the Woopo wetland in South Korea [62]. Some species of Lysinibacillus are positive for catalase and have broad spec- trum antimicrobial activity [63]. Rummeliibacillu spycnus was recently shown to produce an arginine metabolic enzyme with high activity [64] and was speculated to promote better argi-

nine absorption by tilapia on a woody diet in this study. Interestingly, Microbacterium foliorum, which produces a cold-active α-amylase [65], was observed in the guts of tilapia fed fermented MOL leaf meal (Table 3). The limited use of carbohydrates in the diet of fish may Table 4. The dissimilarity of bacteria community associated with different feed groups. C MOL FMOL FM FFM C / 76.50% 87.50% 63.60% 84.60% MOL 76.50% / 68.40% 71.40% 62.50% FMOL 87.50% 68.40% / 69.20% 86.70% FM 63.60% 71.40% 69.20% / 100% FFM 84.60% 62.50% 86.70% 100% / The five groups respectively are control group (C), Moringa oleifera Lam group (MOL), fermented Moringa oleifera Lam group (FMOL), Folium mori leaf meal group (FM) and fermented Folium mori leaf meal group (FFM). https://doi.org/10.1371/journal.pone.0235560.t004 PLOS ONE Effects of wood forages on gut bacterial biodiversity and bioactivity in tilapia PLOS ONE | https://doi.org/10.1371/journal.pone.0235560 July 2, 2020 11 / 17