Hui Shen

Objective: The highly lethal Vibrio disease (HLVD) caused by highly lethal Vibrio parahaemolyticus (VpHLVD) represents a significant threat to Pacific white shrimp Litopenaeus vannamei aquaculture. The existing disease control approaches often rely on antibiotics, leading to drug resistance and environmental pollution, thereby limiting the sustainable development of shrimp farming. Probiotics are recognized for their ability to enhance host disease resistance and to inhibit pathogenic bacteria with high safety and environmental friendliness. Methods: We evaluated the potential of a probiotic bacterial dietary supplement for HLVD prevention. Bacillus altitudinis strain JSHY-074, isolated from coastal aquaculture environments in Jiangsu, China, was identified based on morphological characterization, 16S ribosomal DNA sequencing, and whole-genome analysis. Its antagonistic activity against V. parahaemolyticus strain SHY1669 was determined by co-culture inhibition tests, and the probiotic effect was verified through a VpHLVD challenge experiment on Pacific white shrimp fed with JSHY-074. Results: The strain JSHY-074 exhibited potent antagonistic activity against V. parahaemolyticus strain SHY1669, with inhibition zones expanding from 12 ± 0.31 mm (mean ± SD) at 6 h of co-culture to 30 ± 0.84 mm at 96 h of co-culture. At an inoculum density of 1.0 × 108 CFU/mL, the inhibition rate reached 88.58% at 72 h. Whole-­genome analysis revealed nine gene clusters associated with secondary metabolite production, with lichenysin preliminarily identified as the primary active compound. The VpHLVD challenge experiment demonstrated that feeding Pacific white shrimp with strain JSHY-074 achieved a 70% survival rate within 5 d and a 5,942-fold reduction in hepatopancreas pathogen load (from 6.12 × 107 to 1.03 × 104 copies/ng). Conclusions: This is the first report of B. altitudinis exhibiting antagonistic activity against VpHLVD. The high technological readiness level of B. altitudinis JSHY-074 makes it a versatile probiotic feed additive, and its safety and effectiveness make it well suited for fast deployment and use in shrimp farming, offering a nutritional approach to reducing antibiotic dependency and promoting sustainable aquaculture practices.

Ecytonucleospora hepatopenaei (EHP) is an obligate intracellular microsporidian that causes severe growth retardation in Penaeus vannamei (P. vannamei ), yet its pathogenic mechanisms remain incompletely clear. As the spore wall is essential for resisting environmental stress and mediating host invasion, characterising its protein components is critical for elucidating EHP biology. In this study, we identified and cloned a novel spore wall protein, EhSWP26. Sequence analysis revealed that EhSWP26 contains a 687 bp open reading frame encoding a 228‐amino‐acid polypeptide, lacking a signal peptide and transmembrane domain but harbouring a conserved MICSWaP (Microsporidia Spore Wall and Associated Proteins) domain characteristic of the microsporidian MICSWaPs family. Immunoelectron microscopy and indirect immunofluorescence assays demonstrated that EhSWP26 localises to the mature spore wall, where it is distributed in both the exospore and endospore layers. RNA interference further confirmed that silencing EhSWP26 significantly reduced its transcript level and led to a marked decrease in EHP spore load in shrimp hepatopancreas. Collectively, these findings indicate that EhSWP26 is a key structural and functional component associated with spore wall formation and host infection, likely playing an essential role in the EHP life cycle. This study enhances our understanding of EHP spore wall composition and function, provides a foundation for investigating its proliferation and infection mechanisms, and highlights EhSWP26 as a promising target for diagnostic development and therapeutic intervention in EHP disease of P. vannamei .