Integrated Toxicological and Immunological Evaluation of Rhizophora apiculata Leaf Extract Against Aeromonas hydrophila Infection in Zebrafish (Danio rerio).

The increasing prevalence of antimicrobial resistance in aquaculture has stimulated interest in plant-derived alternatives for bacterial disease management; however, their antimicrobial efficacy must be evaluated alongside host toxicity and biological responses. In the present study, the antibacterial, antibiofilm, toxicological, histopathological, and immunomodulatory effects of Rhizophora apiculata leaf extract were investigated against Aeromonas hydrophila using zebrafish (Danio rerio) as a vertebrate model. The methanolic leaf extract was characterized by FTIR and GC-MS analyses, which indicated the presence of diverse functional groups and putatively identified metabolites belonging to several chemical classes. The extract exhibited antibacterial activity against A. hydrophila, with a minimum inhibitory concentration (MIC) of 125 μg/mL, compared with 15.6 μg/mL for ampicillin. Quantitative crystal violet and confocal microscopy analyses demonstrated concentration- and time-dependent inhibition and disruption of established biofilms, with the greatest effect observed at 500 μg/mL after 48 h. Acute toxicity assessment revealed concentration-dependent mortality, with 96-h LC50 values of 375 μg/mL for embryos and larvae and 200.5 μg/mL for adults, indicating greater sensitivity of adult fish. In A. hydrophila-challenged zebrafish, treatment with the extract reduced infection-associated gill and muscle alterations and improved the observed developmental morphology. The treatment was also associated with differential expression of IL-8, TNF-α, and TGF-β, indicating modulation of immune-related transcriptional responses. However, the treatment concentration of 250 μg/mL exceeded the estimated adult LC50, limiting interpretation of these protective responses as evidence of a safe therapeutic dose. Overall, the findings demonstrate antibacterial, antibiofilm, tissue-protective, and transcriptional immunomodulatory activity of R. apiculata extract, while emphasizing the need for lower-dose studies to establish a safer efficacy-toxicity window for potential aquaculture applications.