Abstract
INTRODUCTION: The hepatopancreas of Litopenaeus vannamei plays a central role in digestion, metabolism, mineral homeostasis, and immune defense; however, its strain-specific responsiveness to probiotics remains insufficiently characterized. This study aimed to elucidate the comparative effects of a single-strain probiotic (Lactobacillus salivarius GZPH2; HH) and a mixed-strain consortium (EM; TH) on hepatopancreatic function under tropical semi-intensive culture conditions. METHODS: An integrated multi-omics approach, combining histology, mineral profiling, 16S/18S rRNA sequencing, and 4D data-independent acquisition (4D-DIA) proteomics, was applied to evaluate probiotic-induced changes after 90 days of feeding, with a non-supplemented group (WH) as control. RESULTS: Both probiotics significantly improved growth, survival, and feed efficiency, increasing biomass by 26-27% relative to the control; however, distinct mechanistic responses were observed. HH enhanced hepatopancreatic regeneration by increasing embryonic (E) and fibrillar (F) cells while reducing blister-like (B) and resorptive (R) cells, alongside greater accumulation of Mg, Fe, Ca, and Se. It also promoted microbial evenness and enriched beneficial Alphaproteobacteria (e.g., Labrenzia, Tropicibacter) and fungal taxa (Candida-Lodderomyces clade). Proteomic analysis revealed upregulation of carbohydrate metabolism, calcium regulation, immune-related proteins, and antioxidant enzymes, including hemocyanin, crustin-like proteins, chitinase, and catalase. In contrast, TH maintained a storage-oriented morphology, exhibited lower mineral deposition and microbial diversity, was dominated by Bacillus, and preferentially enriched proteolytic enzymes and redox-related pathways. DISCUSSION: These findings demonstrate that the single-strain probiotic GZPH2 induced a more regenerative, metabolically efficient, and immunologically robust hepatopancreatic state than the mixed consortium. These findings provide multi-omics evidence supporting strain-specific probiotic selection as a precision strategy to enhance shrimp health and sustainability in aquaculture.