Abstract

Translucent post-larvae disease (TPD) is an emerging shrimp infectious disease caused by the highly lethal Vibrio parahaemolyticus ( Vp TPD), which has caused severe mortality and significant economic losses in shrimp aquaculture. Understanding the pathogenic mechanisms of Vp TPD is essential for developing effective strategies to prevent and control TPD. Outer membrane vesicles (OMVs) act as delivery vehicles for virulence factors in Gram-negative bacteria, and play central roles in host-pathogen interactions. However, their specific contributions to the pathogenicity of highly virulent Vibrio strains remain poorly understood. In this study, OMVs were isolated from Vp TPD ( Vp TPD-OMVs) using differential ultracentrifugation. Their typical spherical morphology and intact membrane structure were confirmed through multiple characterization methods. Proteomic analysis based on Nano-HPLC-MS/MS identified 846 OMV-shuttled proteins, with significant enrichment of outer membrane proteins, adhesins, toxins, secretion system components, immunomodulatory proteins, and iron uptake-related proteins. Notably, three VHVP-associated virulence factors were all identified and further validated in Vp TPD-OMVs using dot blot analysis. Fluorescently labeled OMVs were shown to be internalized by shrimp hemocytes in vitro . Further in vivo injection experiments showed that Vp TPD-OMVs induced the significant upregulation of TLR and its downstream transcription factor Relish , as well as increased expression of the mitochondrial quality-control-related genes Pink1 and Parkin . Moreover, the response was accompanied by the significant downregulation of the stress marker JNK and the vesicle trafficking gene Rab6a . Collectively, this study confirm that Vp TPD-OMVs carry multiple proteins and can modulate host gene expression, highlighting their role as key mediators of Vp TPD pathogenicity and providing a new perspective for elucidating the molecular mechanisms under