Integrated metabolome-SNP-gut microbiome analysis deciphers the molecular basis of EHP resistance in Penaeus vannamei.

Enterocytozoon hepatopenaei (EHP) has emerged as a highly virulent pathogen in shrimp aquaculture, yet the molecular determinants governing host resistance remain incompletely characterized. This study employed an integrated multi-omics strategy (metabolome, transcriptome, and microbiome) to elucidate the molecular basis of EHP resistance in Penaeus vannamei. Comparative metabolomic profiling of EHP-resistant (R4029) and susceptible (S4104) family lines identified 1614 metabolites, primarily organic acids (35.07 %) and lipids (21.56 %). Orthogonal partial least squares discriminant analysis (OPLS-DA) revealed significant intergroup metabolic differentiation (Q2 > 0.9), with 428 and 388 metabolites exhibiting differential abundance (variable importance in projection |VIP| > 1, false discovery rate-adjusted P < 0.05) identified in uninfected (S4104-D vs R4029-D) and infected (S4104-G vs R4029-G) states, respectively. Joint KEGG pathway enrichment analysis of transcriptomic and metabolomic datasets identified 661 commonly dysregulated genes (DEGs) and 205 differentially abundant metabolites (DEMs), with glycine/serine/threonine metabolism (ko00260) and cysteine/methionine metabolism (ko00270) emerging as pivotal pathways underpining resistance. Integrative multi-omics analysis using O2PLS modelling revealed robust correlations between 12 key DEGs and 15 DEMs, thereby prioritizing macrophage mannose receptor 1 (MRC1) and betaine-homocysteine S-methyltransferase (BHMT) as principal candidate genes. Single-nucleotide polymorphism (SNP) screening identified seven BHMT sequence variants, including three variants significantly associated with resistance (BHMT-8159 in intron 3; BHMT-12167 and BHMT-12710 in exonic regions), as well as fourteen MRC1 variants, of which six showed significant associations (P < 0.05), most notably the missense mutation MRC1-759 (A > C) inducing a Thr253Pro substitution (full-length MRC1 sequence) that alters protein secondary structure. Gut microbiome characterization demonstrated elevated α-diversity (P < 0.05) and enhanced community stability in resistant shrimp, with selective enrichment of putatively beneficial taxa (Photobacterium, Ralstonia). Collectively, these findings suggest that EHP resistance is mediated by coordinated transcriptional and metabolic reprogramming, functionally consequential genetic polymorphisms, and stable microbial community dynamics, thereby establishing a multi-omics framework for the selective breeding of EHP-resistant shrimp.