Identification of LysC as a key factor in shrimp intestinal immunity.
(A) Venn diagram of the overlap of two sets of differentially expressed genes. Transcriptome sequencing was performed to screen the genes whose expression was induced after V. anguillarum oral challenge (12 h after challenge) and suppressed after antibiotic feeding (3 d after the treatment). Each group consisted of at least 30 animals. Three biological replicates were performed. Only the genes with an FPKM ≥ 2 in the control group were considered as valid. Differential expression was accepted with a fold change ≥ 2 and an adjusted p value ≤ 0.05. (B) Heat map showing the expression level of differentially expressed genes. The mean of the average FPKM value from three biological replicates of each group for each gene were shown after taking the logarithm. LysC, C-type lysozyme; Ldl receptor, low-density lipoprotein receptor; Ubc E2, ubiquitin-conjugating enzyme E2 R2; Ppaf2, phenoloxidase-activating factor 2. (C) Temporal expression profiles of LysC protein after oral challenge. Bacterial suspension (5 × 106 CFU) was introduced orally into the shrimp intestine. LysC protein level in intestine was detected at indicated time after challenge, with β-actin as the internal reference. (D) Expression level of the LysC protein after antibiotic treatment and subsequent V. anguillarum challenge. The mixture of antibiotics was introduced into shrimp intestine, with water as control. Bacteria were delivered into intestine 3 d later. LysC level was detected 3 d after antibiotics treatment or 12 h after bacterial infection. The western blotting images are representative of three independent replicates. Gray values were analyzed using ImageJ software. Each sample originated from at least five shrimp.