Abstract
Vibrio parahaemolyticus causes mass mortality in global penaeid shrimp aquaculture worldwide, with lethality exceeding 90 %, threatening global food security and economic sustainability. As alternatives to antibiotics, bacteriophages and their lytic enzymes offer target specificity, minimal resistance development, and high bactericidal efficiency. In this study, ten phages were isolated from Litopenaeus vannamei aquaculture ponds and adjacent estuarine areas using Vp499 as the host, which were isolated by our lab. Among these, phage SHY-Vp8 exhibited the highest titer against the host Vp499.Its optimal multiplicity of infection (MOI) was determined to be 1. Notably, SHY-Vp8 demonstrated a superior lytic capacity, with a high burst size (96 PFU/cell) and a short latent period (30 min), representing an 18-41 % improvement in lytic efficiency over previously reported Vibrio phages. Furthermore, it exhibited exceptional environmental resilience, tolerating temperatures up to 60 °C and a broad pH range (3-12), surpassing the stability thresholds of most known vibriophages. Whole-genome sequencing indicated a double-stranded DNA genome of 58,525 bp with 46.38 % GC content. Bioinformatic annotation identified 85 predicted genes, of which 31 encoded functionally characterized proteins. No tRNA or virulence genes were detected, demonstrating potential for therapeutic applications in aquaculture. Transmission electron microscopy confirmed an icosahedral capsid and a long non-contractile tail, classifying SHY-Vp8 within the Siphoviridae family. Notably, the gp59 gene was predicted to encode an endolysin. The gene was amplified cloned and expressed. The lytic activity of Lys59 exhibited a concentration-dependent increase, with peak activity observed at 50 μg/mL. Lys59 alone was capable of lysing Vp499 without the aid of EDTA; however, pretreatment with EDTA significantly enhanced its lytic efficiency. Results indicate that both SHY-Vp8 and Lys59 exhibit promising potential for controlling V. parahaemolyticus infections in L. vannamei, offering a novel and sustainable strategy for disease management in aquaculture-particularly in pond water treatment and seafood safety enhancement. These excellent in vitro activities and characteristics provide a solid foundation for further development of in vivo infection models and eco-friendly biocontrol agents.