Transcriptome-Based Analysis of the Effects of Salt Stress on the Embryos of Hypomesus nipponensis

The increasing salinization and alkalization of inland waters in China pose a growing threat to the early developmental stages of fish. This study investigated the survival rates and transcriptomic response mechanisms of pond smelt (Hypomesus nipponensis) embryos, a species naturally adapted to brackish water, under different salinity stresses. Fertilized eggs were exposed to four salinity gradients: freshwater (0‱, experimental control) and 7‱, 14‱, and 21‱ for 7 days. The results showed that the upper salinity tolerance limit for embryos was between 21‱ and 28‱; hatching rates were zero in the 28‱ and 35‱ groups, and the hatching rate in the 21‱ group decreased to 40.3 ± 6.1%. Transcriptome sequencing analysis of the 0‱, 7‱, 14‱, and 21‱ groups revealed that salinity stress induced extensive differential gene expression in a non-monotonic response pattern. In the 7‱ and 14‱ groups, the number of downregulated genes (approximately 10,000) was significantly higher than that of upregulated genes (approximately 3300). In contrast, in the high-salinity 21‱ group, upregulated genes increased to 4535. Functional enrichment analysis revealed that the calcium signaling pathway, axon guidance, and glutamatergic synapse pathway were significantly enriched across all salinity groups. Changes in the expression of key differential genes (e.g., sparc, hspa4a, aldob, and csmd1a) further confirmed molecular events such as dysregulation of calcium homeostasis, stress protein activation, metabolic inhibition, and neurodevelopmental regulation. The qRT-PCR validation results were highly consistent with the transcriptome data. This study is the first to systematically reveal the molecular regulatory network of pond smelt embryos in response to increasing salinity relative to a freshwater reference point. Our findings indicate their potential for proliferation in moderate saline-alkaline waters (≤14‱), while high salinity (≥21‱) significantly reduces hatching success and triggers a strong stress response. These results provide an important molecular basis and theoretical reference for the enhancement and aquaculture of this species in saline-alkaline waters.