Global warming poses a significant threat to aquaculture, particularly for cold-water species like rainbow trout (Oncorhynchus mykiss). Understanding the molecular mechanisms underlying stress responses is crucial for developing resilient strains. This study investigates the dual stress of salinity and temperature response of “Shuike No. 1” (SK), a pioneering commercially bred rainbow trout strain in China, using RNA-sequencing of gill, intestine, and liver tissues from fish exposed to four treatment combinations: freshwater at 16 °C, freshwater at 25 °C, saltwater (30‰) at 16 °C, and saltwater at 25 °C. Differential gene expression analysis identified a substantial number of DEGs, with the liver showing the most pronounced response and a clear synergistic effect observed under combined high-temperature and salinity stress. Weighted gene co-expression network analysis (WGCNA) revealed stress-responsive gene modules and identified hub genes, primarily associated with gene expression, endoplasmic reticulum (ER) function, disease immunity, energy metabolism, and substance transport. Key hub genes included klf9, fkbp5a, fkbp5b, ef2, cirbp, atp1b1, atp1b2, foxi3b, smoc1, and arf1. Functional enrichment analysis confirmed the prominent role of ER stress, particularly the pathway “protein processing in the endoplasmic reticulum.” Our results reveal complex, tissue-specific responses to dual stress, with high temperature exerting a stronger influence than salinity. These findings provide valuable insights into the molecular mechanisms underpinning dual stress responses in SK, informing future breeding programs for enhanced resilience in the face of climate change.

High-throughput transcriptomic analyses have identified numerous candidate miRNA–mRNA and long non-coding RNA (lncRNA) regulatory networks in teleosts, but most remain without systematic functional validation or mechanistic definition. Here, by interrogating miRNA–lncRNA networks in rainbow trout (Oncorhynchus mykiss) gonads, we define their roles in meiotic progression and gonadal development. From preliminary screening, we identified lncRNA74687 as a central node and characterised its function. Subcellular localisation showed predominant nuclear enrichment of lncRNA74687 in gonadal cells. Dual-luciferase assays confirmed miR-15a-5p targeting of Cyclin E (CCNE1) and lncRNA74687. Functional studies showed that concurrent overexpression of lncRNA74687 and inhibition of miR-15a-5p synergistically increased the CCNE1 protein to maximal levels. 5-ethynyl-2′-deoxyuridine (EdU) assays showed that knockdown of lncRNA74687 and CCNE1 in rainbow trout gonadal (RTG-2) cells reduced proliferation by 36.4% and 41.2%, respectively (p < 0.05). Immunofluorescence indicated that lncRNA74687 increased Synaptonemal Complex Protein 1 (SYCP1) signalling 6.93-fold in gonadal cells via CCNE1. In vivo, lncRNA74687 knockdown increased miR-15a-5p expression 6.34-fold relative to the wild-type controls (p < 0.01). Transcriptomic profiling revealed broad downregulation of meiosis-related genes in lncRNA74687-deficient gonads, with the strongest reduction in mstrg1 expression, indicating a key role of lncRNA74687 in germ-cell meiotic progression. Together, these data show that lncRNA74687 enhances CCNE1 mRNA and the CCNE1 protein in rainbow trout by competitively binding miR-15a-5p. This lncRNA74687–miR-15a-5p–CCNE1 axis regulates gonadal cell proliferation and meiotic gene expression during gonadal development.

As a commercially valuable aquaculture species, rainbow trout (Oncorhynchus mykiss) urgently require solutions to growth inhibition associated with reproductive development. To elucidate the function of the cell cycle regulator Cyclin E genes (CCNE1 and CCNE2) in this process, we cloned the genes and analyzed their relative expression across various tissues and gonadal developmental stages. Using RNA interference (RNAi) and overexpression in RTG2 cells, we examined the effects of CCNE on cell viability, proliferation, and meiotic gene expression. Results showed that the open reading frame lengths of CCNE1 and CCNE2 were 1230 bp and 1188 bp, encoding 408 and 395 amino acids, respectively. Both proteins contain two conserved cyclin boxes, exhibit high structural similarity, and are phylogenetically most closely related to Oncorhynchus tshawytscha and Oncorhynchus kisutch. Expression and localization analyses revealed that CCNE1 was highly expressed in the ovary, while CCNE2 was highly expressed in the testis. Both proteins were expressed during fertilized egg development and key gonadal stages (at 13, 21, and 35 months post-fertilization). CCNE expression positively correlated with RTG2 cell viability and proliferation, with immunofluorescence confirming that CCNE is localized in the nucleus. Knockdown or overexpression of CCNE induced the differential expression of reproductive-related genes and key meiotic regulators. These findings suggest that CCNE1 and CCNE2 balance meiosis and gamete development through specific regulatory mechanisms, and their dysregulation may be a key factor underlying meiosis inhibition and reproductive development abnormalities.

Rainbow trout (Oncorhynchus mykiss) is an economically important fish species, in which the muscle nutritional composition of market-size fish can vary significantly due to differences in genetic background and breeding environments. This study employed a metabolomics approach to conduct an in-depth comparative analysis of muscle samples from our independently bred new variety “All-Female No. 1”, as well as Spanish and Danish all-female strains. A total of 2198 metabolites were identified. Following screening with PCA and PLS-DA, 228 differential metabolites were obtained. These were most abundantly enriched in the categories of organic acids and derivatives, benzene and substituted derivatives, and amino acids and metabolites. KEGG pathway enrichment analysis revealed that the differential metabolites had the most significant impact on the phenylalanine, tyrosine, and tryptophan biosynthesis pathway. Further targeted quantification of amino acid metabolites identified 11 differentially expressed amino acids, which also exerted the strongest influence on this key pathway. Consequently, L-tyrosine, tryptamine, and L-phenylalanine were determined to be the key metabolites affecting the phenylalanine, tyrosine, and tryptophan biosynthesis pathway in the muscle of rainbow trout from different germplasms. This study provides a comprehensive evaluation of the nutritional value, particularly regarding amino acid profiles, of rainbow trout from different genetic sources, offering critical data and a scientific basis for subsequent related research.

Background/Objectives: The level of stocking density serves as a critical modulator of both growth performance and physiological homeostasis in fish reared within recirculating aquaculture systems (RAS). Methods: In this study, an 8-week feeding trial was designed incorporating three density gradients specifically 30, 50, and 70 kg/m³ with the aim of systematically investigating the consequent alterations in growth rates, digestive functionality, antioxidative status, and hepatic transcriptional profiles of triploid rainbow trout (Oncorhynchus mykiss). Results: All treatment groups maintained their growth vigor, and there were no significant differences in final body weight or body length. The final stocking densities were 36.85 ± 0.73, 59.70 ± 2.73, and 70.97 ± 0.32 kg/m³ in the 30G, 50G, and 70G groups, respectively. However, survival rates decreased as stocking density increased. Digestive enzyme activities were generally higher at 50 kg/m³ but declined at 70 kg/m³, particularly intestinal amylase and trypsin activities. Hepatic MDA content increased with stocking density, reaching 5.47 ± 0.28 nmol/mg protein at 70 kg/m³, while T-AOC decreased significantly to 25.00 ± 1.52 μmol/g protein (p < 0.05), indicating enhanced oxidative stress and weakened antioxidant defense. Mild hepatocellular vacuolation was also observed at higher stocking densities. Transcriptomic analysis identified 558 differentially expressed genes between the 30 kg/m³ group and the 70 kg/m³ group; these genes were primarily enriched in pathways related to foreign body metabolism, ferroptosis, and cell adhesion molecules (p < 0.05). At 70 kg/m³, several antioxidant-related genes, including cat, gclc, gsr, gpx1, gpx4a, prdx1, and hsp70, were downregulated, whereas sod1, sod2, maf, me1, and gadd45a were upregulated. Conclusions: These results indicate that rainbow trout can maintain growth at stocking densities up to 70 kg/m³ under the tested RAS conditions, but higher density is associated with reduced digestive capacity and increasing hepatic oxidative stress. The 50 kg/m³ treatment showed a relatively favorable balance between growth, digestive function, and antioxidant status, suggesting its potential suitability for intensive RAS production.