Efficacy of synbiotic diet incorporating Lactobacillus plantarum L20 and Sargassum polycystum for Penaeus monodon against acute hepatopancreatic necrosis disease

Acute hepatopancreatic necrosis disease (AHPND) has inflicted massive economic losses and posed serious threat to the development of penaeid shrimp aquaculture in Malaysia. Although antibiotics were applied for combating AHPND, but they caused antibiotic resistance emergence which directly led to regulate antibiotic restriction for aquaculture in Malaysia. Thus, the alternatives, including Lactobacillus species as probiotic, Sargassum polycystum as prebiotic and their combination as synbiotic were introduced against AHPND outbreak. In the initial study, several Lactobacillus strains that possessed in vitro anti-Vibrio properties were identified and screened from lactic acid bacteria (LAB) isolates of different marketed food fish using biochemical characterization, 16S ribosomal RNA gene identification and Vibrio antagonistic analyses. Then, the 108 CFU/mL of these single Lactobacillus strains supplemented diets were used for 35 days of feeding trial using Penaeus monodon postlarvae, followed by immersion challenge using 6 × 105 CFU/mL of AHPND-causing Vibrio parahaemolyticus S2-4. An effective Lactobacillus strain that possessed in vivo anti-AHPND was chosen upon analyses of growth performance, survival, bacterial enumeration and histopathology. In the second study, the effective 108 CFU/mL of L. plantarum L20 (probiotic), 2 % of S. polycystum (prebiotic) and their combination (synbiotic) supplemented diets were used for the feeding trial and challenge test using same regime of the initial study. The most effective supplemented diet was chosen upon analyses of growth performance, survival, bacterial enumeration, immune gene expression and histopathology. For the final study, the effective synbiotic diet fed shrimp was subjected to gastrointestinal microbiota analyses of high throughput 16S rRNA amplicon sequencing and histopathology. In the result of initial study, L. plantarum L20 showed significant (p<0.05) highest of in vitro anti-Vibrio activities, high shrimp growth rate (5.32 ± 0.29 %), high L20 abdomen colonization (23.676 ± 0.193 log10 CFU/mL), high post-challenge survival (80 ± 4.71 %), lowest S2-4 cephalothorax colonization (4.504 ± 0.203 Log10 CFU/mL) and hepatocytes with epithelial cells protection. Thus, L20 was chosen as an effective probiotic among other strains for subsequent second study. In the result of second study, synbiotic diet showed significant (p<0.05) highest shrimp growth rate (3.94 ± 0.33 %), high L20 abdomen colonization (3.676 ± 0.193 log10 CFU/mL), high post-challenge survival (76.67 ± 4.71 %), lowest S2-4 cephalothorax colonization (4.504 ± 0.203 Log10 CFU/mL), hepatocytes with epithelial cells protection, improve shrimp cephalothorax immunity of feeding trial including cell-adhesive signaling (at 2 fold up-regulation), prophenoloxidase system (at 3 fold up- regulation) with lysozyme secretion (at 5 fold up-regulation) and challenge test including prophenoloxidase system (at 2 fold up-regulation), toll-like signaling (at 2 fold up-regulation) with penaeidin secretion (at 3 fold up-regulation). Thus, the synbiotic diet was considered as the best supplement diet compared with probiotic and prebiotic diets. In result of last study, the synbiotic diet could improve shrimp gastrointestinal microbiota as significant (p<0.05) increased Lactobacillaceae, Mycoplasmataceae, and Devosiaceae with decreased Vibrionaceae, enriched potential microbial glycolysis and gastrointestinal tissue protection. This study suggested the synbiotic improve growth performance, immune responses, gastrointestinal microbiota and disease resistance of shrimp. Therefore, it can be applied a sustainable solution to prevent substantial production losses in shrimp aquaculture.

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