Single-nucleus RNA sequencing reveals regulatory mechanisms of bile acids on lipid metabolism in Litopenaeus vannamei

During lipid metabolism, bile acids are involved in the emulsification and absorption of lipids, and the liver serves as the primary organ responsible for mediating their regulatory functions. Crustaceans lack de novo bile acid synthesis and may differ substantially from vertebrates in bile acid metabolism. To investigate the role of bile acids in the lipid metabolism of crustaceans, this study divided 480 Litopenaeus vannamei (initial weight: 0.640 ± 0.003 g) into three groups. Each group included four replicate tanks, with 40 shrimp per tank. Fed the following diets for an 8-week feeding trial, respectively: high fishmeal diet (HF, 25% fish meal), low fishmeal diet (LF, 12.5% fish meal), or low fishmeal diet supplemented with 400 mg/kg bile acid (LFB). The final body weight, weight gain rate and average daily gain of the LFB group were significantly higher than those of the LF and HF groups ( P < 0.05). Hepatopancreatic histomorphological and biochemical analyses revealed that the bile acid supplementation alleviated the accumulation of collagen fibers in the hepatopancreas and reduced the activity of glutamic-pyruvic transaminase (GPT) ( P < 0.001). Single-nucleus RNA sequencing (snRNA-Seq) enabled the construction of the first comprehensive hepatopancreatic cell atlas in invertebrates, with 11 major cell types identified via transcriptomic profiling. Bile acid administration significantly increased F cell abundance while reducing the number of R cell and B cell populations ( P < 0.05). Intercellular communication analysis demonstrated that bile acid supplementation decreased signal enrichment in neural cell adhesion molecule (NCAM) and collagen (COLLAGEN) pathways, with differential functional pathways between the LFB group and LF group predominantly enriched in non-alcoholic fatty liver disease (NAFLD) pathways. Notably, Maribacter and Tamlana emerged as significantly differentiated genera in the bile acid treatment group, exhibiting functional associations with the mitigation of NAFLD and the catalysis of short-chain fatty acid degradation. Pseudotime trajectory analysis further uncovered potential hepatopancreatic cell differentiation pathways: E cells, as progenitor cells, differentiate into R cells, which subsequently bifurcate into B cell and F cell lineages. Collectively, bile acids alleviate hepatopancreatic fibrosis and inflammation by inhibiting cell junction-related signaling pathways, while concurrently regulating lipid metabolism through the enhancement of F cell proportions.