Transcriptome analysis revealed immune protection conferred by bath vaccination with formalin-killed Vibrio...

Acute hepatopancreatic necrosis disease (AHPND) remains a challenge in shrimp aquaculture. Growing interest in the adaptive-like immunity of crustaceans has raised prospects for vaccination strategies. In this study, we utilized formalin-killed Vibrio parahaemolyticus (FKC-Vp) strains (TUMSAT_N3, TUMSAT_FP11, and TUMSAT_D6) as vaccine administered via bath vaccination in Penaeus vannamei shrimp. The FKC-Vp strains and efficient bath conditions were initially optimized. Shrimps were immersed for either 1 h or 2 h at 1% or 2% FKC concentration and then transferred to new tanks for subsequent observation. Among which, shrimps bathed with 1% FKC-VpFP11 for 1 h showed higher survival rate and upregulated expression of immune-related genes without histopathological damage in the hepatopancreas. Based on these optimal conditions, RNA sequencing via Illumina platform was then performed on hepatopancreas and stomach tissues collected at 24 h post-vaccination (pv). Transcriptomic profile revealed differentially expressed genes (DEGs), where GO and KEGG analyses further identified enriched metabolic, stress response, and bacterial mitigation signaling pathways. Twelve significant immune-related DEGs involved in pathogen recognition, antimicrobial defense, ROS elimination, and epigenetic modulation were further validated via qPCR. These DEGs were examined at 24 h and 7 d pv, and 24 h post-challenge (pc). Results showed temporal expression shift from an acute-phase response at 24 h pv, to a primed state at 7 d pv, followed by an activated defense state at 24 h pc. Notably, antimicrobial effectors (GILT, hemocyanin C-chain) peaked at 24 h pv, while recognition factors (C-type lectin, perlucin) and putative epigenetic regulators (zinc finger, E3 ubiquitin ligase, late histone H1) remained sustained or upregulated through 7 d pv. A significant interaction between time and vaccinated treatment was observed in GILT. Despite the Vp_AHPND challenge occurring 7 d pv, all DEGs were upregulated at 24 h pc, with significant elevated expression in hemocyanin C-chain. This correlated with increased survival rate (80%) in the vaccinated shrimp compared to the control. Our findings provide a molecular basis for immune priming induced by 1% FKC-VpFP11, suggesting a memory-like mechanism that warrants further investigation. Overall, this study emphasized a cost-effective and protective disease-control strategy for P. vannamei against Vp_AHPND.