A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Aquaculture and Aquatic Resources Management

Thesis (Ph.D.) - Asian Institute of Technology, 2019

The effects of selected plant extracts, guava (Psidium guajava; PGLE), star gooseberry (Phyllanthus acidus PALE), and the mixture of PGLE and PALE on the gastrointestinal microflora, immuno-hematological responses and disease resistance of Nile tilapia (Oreochromis niloticus) challenged with Aeromonas hydrophila and Streptococcus agalactiae were investigated in this study. The plant extracts, hereafter termed phytobiotics, were added to commercial tilapia feed (Charoen Pokphand (CP)-771O) at the rate of 5 and 109 kg" respectively. The first part of the study dealt with evaluating the phytochemical and total phenolic content (TPC) , antioxidant [DPPH (1, 1-diphenyl-2-picryl hydrazyl), ABTS: 2,2'-azino- bis (3-ethylbenzothiazoline-6-sulphonic acid) and FRAP (Ferric reducing antioxidant power assay)] and antimicrobial activities of different solvent leaf extracts of PGLE, PALE and a mixture of PGLE & PALE (Mixed). The phytochemical screening of aqueous (AQ) leaf extract of phytobiotics revealed the presence of alkaloids, flavonoids, phenolics, saponins, and tannins. The AQ extract showed high total phenolic content (TPC) in PGLE (173.4.±9.4 mg GAE g-l), PALE (322.1±17.9 mg GAE g-l), and mixed (140.2±7.5 mg GAE g-l) phytobiotics. The methanol (ME) extract of PGLE had significantly lower (p<0.05) ICso value of DPPH (194.83±13.22 ug mL-1) compared to L- ascorbic acid (299.83±5.13 ug mL-1). The AQ & ethanol (ET) extracts of PALE demonstrated significantly lowest (p<0.05) ICso value of ABTS (165.2±2.0 & 177.40±6.58 ug mL-1) compared to quercetin (308.37±19.08 ug mL-1). The AQ, ET and ME extracts of PGLE demonstrated significantly higher (p<0.05) FRAP values (306.30±O.97, 291.49±3.39 and 388.49±25.82 J.lM FeS04 g-\ respectively) in comparison to L-ascorbic acid (281.51±2.66 J.lM FeS04 g-l). The phytobiotic extracts also demonstrated considerable antimicrobial activity against S. agalactiae and A. hydrophila. AQ leaf extracts of phytobiotics showed bactericidal activity against both bacteria with MIC and MBC values lower than 100 ug mL-1 whereas, AQ extracts of mixed phytobiotic exhibited a strong synergistic impact against S. agalactiae and a nearly additive effect against A. hydrophila, respectively. The in vitro results revealed the presence of alkaloids, flavonoids, phenolics, saponins and tannins in the AQ extracts of PGLE, PALE and mixed phytobiotics. These extracts also demonstrated higher total phenolic content, most potent antioxidant activities and higher antimicrobial and bactericidal activities. Hence, they were chosen for the 'in vivo experiments using Nile tilapia. The second study was based on investigating the effects of dietary supplementation of phytobiotics on gut (distal) microbiota of Nile tilapia. Growth and feed utilization parameters were also monitored to evaluate whether the supplementation of phytobiotics had any adverse effect on them. The results showed that the highest total viable count of the digesta sample was observed in PALE-5 treatment. DGGE DNA fingerprinting patterns extracted from the control and phytobiotic treatment-groups of digesta of tilapia after 0 (Initial) and 12 weeks of feeding were clustered into six major groups at 58 % similarity. The total number of phyla and genera from each treatment ranged from 1 to 3 and 4 to 12, respectively. The most diverse intestinal bacterial community was observed in the fish fed diet supplemented with mixed-IO phytobiotics treatment with 12 genera. Also, PGLE-IO phytobiotic treatment had the highest Margalefs Species richness, Shannon-Wiener index, and evenness (5.06, 2.08, and 2.08). The highest Simpson index of diversity was observed in mixed-lO phytobiotics treatment. Seven phyla of bacteria including Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Planctomycetes, Proteobacteria and Verrucomicrobia were represented in the sequences. The most dominant phylum was Fusobacteria accounting for -78.3% (224 clones) of the total clones, and it was the only phylum found in all experimental treatments. Cetobacterium spp. was the most dominant genera observed among all the experimental treatments. The disappearance in the abundance of phylum Verrucomicrobia & Bacteroidetes and also an emergence of phylum Actinobacteria were observed in the phytobiotic treatment-groups when compared to the control. The gut bacterial community from PGLE-I0 was found significantly (Cs <0.60) different (Cs = 0.45) and PALE-lO & Mixed-lO were found marginally (0.60 :S Cs < 0.85) different (Cs = 0.84 and 0.66) in comparison to the control. It was also observed that the inclusion of plant extracts in fish feeds did not have any detrimental effect on the growth performance and feed utilization efficiency of fish. The third study was conducted to investigate the efficacy of aqueous extract of phytobiotics supplemented diets consisting of leaf-extracts of Psidium guajava (PGLE), Phyllanthus acidus (PALE), and the mixture of PGLE and PALE at the rates of 5 and 10 g kg" of the feed of Nile tilapia (Oreochromis niloticus), respectively. Following 12 weeks of feeding, the fish were challenged with Streptococcus agalactiae for 15 days. Immuno-hematological parameters were measured before and after the challenge. The results showed significantly (p<0.05) enhanced innate immune parameters after administration of the plant extracts. Innate immunity parameters were 2 to 3 times higher than the control; PGLE-I0 showed the highest values. The fish fed PGLE-I0 had the highest post-challenge WBC, RBC, Hb, and Hct values compared to the control. The dietary supplementation of plant extracts had a higher survival percentage compared to the control. The fourth study investigated the protective effects of dietary supplementation of the phytobiotics and their potential for enhanced immune response and disease resistance of Nile tilapia against A. hydrophila infection. The results showed that dietary supplementation of PGLE-l 0 activated the immuno-hematological responses and altered the serological parameters of Nile tilapia. A significantly higher survival was observed in the fish fed diet supplemented with phytobiotics treatment-groups than the control group (p<0.05 by log-rank test). The highest relative percentage survival was found in PALE-lO (87.4%) followed by PGLE-5 (83.7%), PGLE-lO, PALE-5, Mixed-lO (78.9%) and Mixed-5 (74.7%), respectively. In conclusion, the in vitro AQ extracts of PGLE, PALE and mixed plants revealed the presence of flavonoids, phenolics, saponins and tannins, higher total phenolic content, most potent antioxidant, higher antimicrobial and bactericidal activities, and a strong synergism against A. hydrophila and S. agalactiae. In vivo trials also proved that the diet enriched with plant extracts had comparatively higher relative percent survival of Nile tilapia against the A. hydrophila and S. agalactiae infection. Addition of 10 g kg-l of guava leaves (PGLE-I0) improved the hematological and immunological parameters of Nile tilapia. The inclusion of plant extracts to fish feeds demonstrated no detrimental effect on growth performance and feed utilization efficiency of tilapia. The most diverse intestinal bacterial community was observed in the fish fed diet supplemented with mixed-lO phytobiotics treatment with 12 genera. The gut bacterial community from PGLE-lO was significantly different, and P ALE-lO and Mixed-lO were marginally different compared to the control. Since the present study showed the positive effects of phytobiotics on the resistance of fish against pathogenic A. hydrophila and S. agalactiae, it reveals their potential to be used efficiently as an alternative prophylactic and antimicrobial agent in Nile tilapia farming.