Using a three-factor orthogonal design, we investigated the transcriptomic and histological responses of triploid rainbow trout (Oncorhynchus mykiss) gills across three salinities (10, 20, and 30 ppt) over 60 days, with temperature and body size as background conditions. Results showed that 20 ppt was associated with extensive transcriptional reprogramming, with 3333 differentially expressed transcripts (DETs), whereas 30 ppt induced only 120 DETs; WGCNA further identified salinity-correlated modules involved in ion transport and ribosome biogenesis. Histological alterations remained within physiological ranges with no salinity-dependent severity gradient, as confirmed by semi-quantitative scoring. These findings demonstrate that triploid rainbow trout gills employ distinct molecular strategies at medium versus high salinity. Within the 10–30 ppt range examined, 20 ppt was the salinity at which transcriptional reprogramming was most active, suggesting that this intermediate salinity warrants further investigation as a potential acclimation stage.

Salinity, temperature and body weight dominate seawater acclimation in rainbow trout (Oncorhynchus mykiss), yet few studies simultaneously explore their main effects and potential correlative interactive patterns in hepatic responses. A 60-day L₉ (3³) orthogonal trial was performed on triploid rainbow trout with three gradients of body weight, temperature and salinity. Hepatic transcriptomics revealed that salinity drove global transcriptional remodeling and high salinity induced far fewer DEGs than medium salinity. WGCNA screened a salinity-positive blue module (r = 0.408, p = 0.0346), while alternative splicing confirmed extensive salinity-dependent post-transcriptional regulation. Semi-quantitative histology showed that 20 °C was associated with more pronounced salinity-caused hepatocellular vacuolation and karyopyknosis in the orthogonal test. Survival statistics indicated that salinity was the only factor with significant main effects (p < 0.05), and the 500 g-10 °C-10 ppt group obtained the highest survival. This multi-omics and histological dataset reveals a suggestive regulatory hierarchy-like pattern: salinity acts as the primary driver, temperature serves as a synergistic amplifier, and body weight plays a minor modulatory role. These findings provide a theoretical basis for developing size-specific salinity acclimation protocols in commercial triploid rainbow trout farming.