eng Oriental Scientific Publishing Company Biosciences Biotechnology Research Asia 0973-1245 2025-12-30 22 4 1755 1772 10.13005/bbra/3474 57228 article Salem Sundarrajan Maithili 1 Muthu Ganesh Boopathi 1 Kandhasamy Lalitha 1 Department of Microbiology, AVS College of Arts and Science (Autonomous), Tamil Nadu, India. Spodoptera litura control is genuinely expected to replace the deadly compound pesticides; the disadvantages of the invention pesticides are issues, an effect on human and animals, environmental pollution and killing the non-target insect and highly resistance response to an insect pest. This situation used as ecologically agreeable, moved nearer to the bio pesticide. In this study, four strains were isolated from the zebra fish gut, among the two strains the highest bacterial growth was observed in ZBF-2 Bacillus subtilis, ZBF-4 Bacillus cereus. In view of biochemical, physiological and molecular characterization the isolated zebra fish gut was distinguished as having a place with Bacillaceae family. Phylogenetic investigation of the 16S rDNA sequence of zebra fish gut bacteria are ZBF-2 Bacillus subtilis, ZBF-4 Bacillus cereus. In Danio rerio gut bacterial optimal growth conditions were tested with different media optimal growth level with ZBF-2 and ZBF-4 showed the highest growth rates under optimized conditions, including glucose as the key carbon source, peptone as the nitrogen source, pH levels of 4 to 7 and a temperature of 35ºC at 72hrs .The two strains, ZBF-2Bacillus subtilis and ZBF-4Bacillus cereus, were selected for 16S rRNA sequencing, and the resulting sequences identified as and get the accession number of Bacillus subtilis (PV297978) and Bacillus cereus (PV300533). These optimized conditions fostered the growth of gut microbes that are important for the secondary metabolites enhance for the bioactive compounds may be responsible for the insecticidal properties.  And we further tested, with the isolated bacterial cell free supernatant of ZBF-2 and ZBF-4 produced 76-80% mortality at 48hrs in Spodopteralitura larvae. This study shows that, the symbiotic bacteria ZBF-2 Bacillus subtilis, ZBF-4 Bacillus cereus are virulent, and their secondary metabolites are also toxic and can be utilized in pest control programs. https://www.biotech-asia.org/vol22no4/isolation-identification-and-media-optimization-of-gut-bacteria-species-from-danio-rerio-zebra-fish-against-the-spodoptera-litura-larvae/ Bacillus cereus; Bacillus subtilis;Gut bacteria; Larval morality; Optimization; Secondary metabolites