AKT pathway plays a pivotal role in OnMBL/CD91a promoting MФ phagocytosis and bacterial clearance. Western blot showed the protein expression (A) and phosphorylation levels (B) of AKT, mTOR, PTEN, and 4EBP-1 in the head kidney that immunized with S. agalactiae after OnMBL RNAi for 24 h. (C and D) Western blot analysis showing protein or phosphorylation levels of the indicated AKT pathway elements in MФ with or without OnMBL stimulation. (E–G) Immunofluorescence analysis showing the phosphorylation levels of p-mTOR (E), p-PTEN (F), and p-4EBP-1 (G) with or without OnMBL stimulation. Scale bar, 5 μm. (H and I) After OnCD91a RNAi for 12 h, the relative mRNA and phosphorylation levels of the indicated molecules in the head kidney were analyzed by qRT-PCR (n = 4) and Western blot at different time points post–bacterial infection. (J) Tilapia individuals were intraperitoneally injected with AKT inhibitor AKT-IN-6 for 2 consecutive days before the separation of head kidney MФ. MФ from inhibitor-treated or untreated tilapia were stimulated by OnMBL for 12 h, and the phosphorylation levels of indicated molecules were detected by Western blot. (K) Tilapia individuals were treated with AKT-IN-6. The head kidney MФ phagocytosing S. agalactiae in the presence or absence of OnMBL were analyzed by flow cytometry. (L) The phagocytic percentage and MFI of MФ (n = 4). (M) After AKT-IN-6 treatment of tilapia, the isolated MФ were incubated with DCFH-DA (2 μmol/L) for 20 min. After washing, the cells were added to OnMBL (5 μg/mL) preincubated S. agalactiae, incubated at room temperature for 1 h, and analyzed by flow cytometry. (N) Kaplan–Meyer survival plot showed the survival percentage of tilapia (25 fish for each group).