Shrimp aquaculture is constrained by opportunistic bacterial pathogens, particularly Vibrio parahaemolyticus, whose proliferation in hatchery systems is shaped by microbial community structure. We evaluated the antagonistic activity and microbiome effects of two native Bacillus-based probiotic consortia (CN5, RS3) applied alone or combined (MIX) in shrimp larval culture water over 30 days, relative to a no-probiotic control. Treatments were assessed using standardized in vitro inhibition assays, 16S rRNA gene (V3–V4) amplicon sequencing, functional inference, and integrative multivariate and structural modeling. All probiotic treatments showed consistently high antagonistic activity against V. parahaemolyticus, whereas the control showed no inhibition. Amplicon profiling indicated treatment-associated microbiome restructuring, with increased Bacillus dominance and reduced relative abundance of Vibrio spp. under probiotic conditions. Multivariate analyses separated probiotic and control groups, and PLS-SEM identified Bacillus dominance as a central driver of antagonistic activity mediated by inferred bioactive functional potential, while water-quality variables had limited direct effects. Probiotics were administered directly to the culture water once daily after routine water exchange to 1 × 10⁶ CFU mL⁻¹ (CN5 or RS3); MIX was applied 1:1 (v/v) at the same total dose.

Shrimp aquaculture is persistently constrained by opportunistic bacterial pathogens, particularly Vibrio parahaemolyticus, whose proliferation in hatchery systems is strongly influenced by microbial community structure. This study evaluated the antagonistic capacity and microbiome-level effects of two native Bacillus-based probiotic consortia (CN5 and RS3), individually and in combination (MIX), in shrimp larval culture water. Over a 30-day experimental period, probiotic treatments were compared with a no-probiotic control using a combination of standardized in vitro inhibition assays, 16S rRNA gene (V3–V4) amplicon sequencing, functional inference, and integrative multivariate and structural modeling. All probiotic treatments exhibited consistently high antagonistic activity against V. parahaemolyticus, whereas the control did not show inhibition. Amplicon-based profiling revealed a clear treatment-associated restructuring of the water microbiome, characterized by increased Bacillus dominance and reduced relative abundance of Vibrio spp. under probiotic conditions. Multivariate analyses demonstrated robust separation between probiotic and control treatments, and partial least squares structural equation modeling identified Bacillus dominance as a central driver of antagonistic activity, mediated through inferred bioactive functional potential. Water-quality variables showed limited direct influence within the modeled framework. Collectively, these results indicate that native probiotic consortia are associated with stable Bacillus-dominated microbial regimes and strong in vitro suppression of V. parahaemolyticus, supporting their potential role as ecological tools for microbial management in shrimp hatchery systems.