Mechanisms underlying host-specific horizontal transfer of shrimp AHPND virulence plasmid: Vibrio immunity and colonization environment.

Acute hepatopancreatic necrosis disease (AHPND) is a highly contagious and lethal shrimp disease caused by Vibrio species harboring the virulence plasmid pAHPND, which encodes pirAB toxin genes. Here, we employed comprehensive comparative genomic and bioinformatic analyses to elucidate the mechanisms that govern the host-specific horizontal transfer of pAHPND. Our analyses revealed that although the majority of Vibrio host strains harboring pAHPND or pAHPND-related plasmids (71%, 27/38) possessed restriction-modification (R-M) systems, these plasmids encode anti-restriction proteins (ArdC, KlcA), suggesting R-M systems alone may be insufficient to block plasmid dissemination. Of the 92 non-AHPND Vibrio strains with CRISPR-Cas systems targeting pAHPND or its related plasmids, 64 specifically targeted the pAHPND plasmid itself. The primary targets within this plasmid were genes involved in fimbriae biosynthesis. Analysis of antimicrobial resistance genes (ARGs) in these CRISPR-positive strains showed that half (46/92) carried more than five ARGs, indicating robust environmental adaptability. In contrast, Vibrio strains that stably maintained pAHPND lacked CRISPR spacers targeting this plasmid and carried fewer ARGs (with 76% of strains harboring no more than seven ARGs). Collectively, these results suggest that the dissemination of pAHPND is shaped by Vibrio immune defenses - particularly CRISPR-Cas systems - and by selective pressures in the colonization environment, including antibiotic use. These findings highlight the evolutionary interplay between bacterial immunity, environmental stress, and virulence plasmid persistence.