| The intestine is the primary site for nutrient digestion and absorption in fish and also serves as a crucial immune organ. Salinity and temperature are key environmental factors that significantly influence fish physiology and play vital roles in their lives. Salinity, as a specific ecological factor in aquatic environments, can markedly affect fish intestinal structure and function, posing a threat to fish health. Temperature is considered a major environmental factor affecting fish physiology; changes in temperature can influence the activity of digestive enzymes, hydrogen peroxide enzymes, and other enzymes in the intestine, as well as the expression of growth-related genes. It can even disrupt the intestinal morphology and induce intestinal cell apoptosis. The combined effects of salinity and temperature changes on fish physiology are often complex and can intensify the physiological burden on fish. The negative impacts of these combined stressors can far exceed the linear superposition of individual stressors, reducing the fish's ability to adapt to each stressor individually and further threatening their health and survival. Alternative splicing (AS), a key mechanism in gene expression regulation that generates diverse transcripts, not only increases protein diversity but also dynamically adjusts gene expression programs, thereby enhancing the adaptability of organisms to environmental stress. In fish, AS has been shown to play a critical role in responses to various environmental stresses, including temperature and salinity changes. Rainbow trout (Oncorhynchus mykiss), as euryhaline fish with both economic and ecological research significance, exist in anadromous and resident forms. Resident rainbow trout live in freshwater environments and thrive at temperatures between 12-18°C. In China, most cultured rainbow trout are of the resident type, and their production has been constrained by inland cold-water resources, remaining around 40,000 tons. Marine farming is a viable strategy to increase rainbow trout production. However, despite their relatively high tolerance to salinity, rainbow trout exhibit significantly reduced intestinal digestive enzyme activity and growth performance in high-salinity environments compared to freshwater. Additionally, global warming has led to increased seawater temperatures, exacerbating the dual stress of salinity and temperature on rainbow trout and intensifying environmental challenges for marine farming. To investigate the interactive effects of high salinity and high temperature on gene expression in the intestine of rainbow trout and their dynamic AS responses, this study conducted an in-depth analysis of RNA-seq data from rainbow trout intestines under high salinity and high temperature stress. The data were preprocessed using methods identical to those in the original study, including data filtering, quality control, and transcript assembly, and gene counts and FPKM values were calculated. Differentially expressed genes (DEGs) were identified in the high salinity stress group (SI), high temperature stress group (TI), and dual high salinity-high temperature stress group (DI), with counts of 339 (SI), 1350 (TI), and 3581 (DI), respectively. Subsequently, based on genes' counts values, the DESeq2 (v1.44.0) R package was employed to construct a generalized linear model (GLM) for interaction effect analysis. The design matrix included main effects of salinity (0/30‰) and temperature (16/25°C), as well as the salinity-temperature interaction term (~ Salinity + Temperature + Salinity:Temperature). A likelihood ratio test (LRT) was used to compare the full model (with interaction terms) and the reduced model (with only main effects: ~ Salinity + Temperature). Low-expression genes (filtered based on total expression, removing genes with counts totaling less than 10 times the sample number) were excluded before model fitting to enhance the accuracy and stability of model construction. The plotDispEsts function was then called to generate a dispersion estimate plot, confirming the reasonableness of the model and the reliability of the data. The interaction analysis results revealed 113 interaction genes, comprising 6 synergistic genes (2 of which were DEGs) and 107 antagonistic genes (51 of which were DEGs). Although interaction genes accounted for only 0.31% of the background genes, they exhibited strong interaction effect intensity. Using the freshwater control group (FI) as a reference, rMATS-turbo software (v4.1.2) was used to detect and analyze AS events in SI, TI, and DI, including five common splicing types: Skipped Exon (SE), Alternative 3' Splice Site (A3SS), Alternative 5' Splice Site (A5SS), Retained Intron (RI), and Mutually Exclusive Exons (MXE). By comparing with FI (freshwater temperature suitable group), AS events were abundant in SI, TI, and DI, comprising 32.33%, 33.96%, and 33.71% of total events, respectively, with a near 1:1:1 ratio. The local composition of SE : A3SS : A5SS : RI : MXE was approximately 29:30:22:14:5, with A3SS and SE being the most frequent. DAS events (FDR < 0.05 and difference with FI > 10%) made up 1.62%, 45.01%, and 53.37% of events in SI, TI, and DI, respectively, showing significant differences. SE : A3SS : A5SS : RI : MXE ratios varied distinctly across conditions: SI (46:11:17:23:3), TI (54:15:20:5:6), and DI (54:13:20:7:6), with SE remaining the most common. Notably, SI exhibited fewer DAS events compared to TI and DI. Further analysis identified 31, 699 (30 DEGs), and 761 (64 DEGs) genes in DAS events for SI, TI, and DI, respectively, with substantial overlap between TI and DI genes. Seven genes were shared DAS genes across all three stresses, displaying diverse AS events. TI and DI DAS genes were significantly enriched in three KEGG pathways (padj < 0.05): Spliceosome (ko03040), Polycomb Repressive Complex (ko03083), and ATP-Dependent Chromatin Remodeling (ko03082), with high gene overlap. Only five antagonistic genes were DAS genes, and DAS events were exclusive to TI and DI. The results of this study indicate that the interactive effects of high salinity and high temperature stress on overall gene expression in the intestine of rainbow trout may be minimal but have significant impacts on specific genes. Additionally, SE events may play an important role in the response of rainbow trout to different environmental stresses, while RI events may have a unique role in the response of fish to salinity stress. These findings reveal the complex mechanisms by which rainbow trout regulate gene expression to cope with environmental stress, providing new insights into their environmental adaptation strategies. This study offers an important theoretical foundation for understanding the environmental adaptation mechanisms of rainbow trout and aids in the further development of marine farming and strain improvement efforts for this species. |
