Supplementary material for Genetic basis of embryo and juvenile physiological responses to salinity changes of embryos and juveniles in freshwater pipefish

This study aimed to describe the adaptation to salinity changes in freshwater pipefish (Hippichthys heptagonus) embryos and juveniles using transcriptome data. Embryos exposed to 30 ppt salinity showed downregulated cell cycle-associated genes, which play an important role in embryonic development. In addition, retinol metabolism, neuroactive receptor interaction, and peroxisome proliferator-activated receptor signaling pathways were significantly upregulated in the embryos. Interestingly, there was no change in the expression of ion transport and energy metabolism genes. In contrast, juveniles exposed to 30 ppt salinity showed upregulated ion transport genes and immune-related signaling pathways, including lysosomes, phagosomes, autophagy, and mitophagy. Additionally, carbohydrate metabolism genes were upregulated, whereas oxidative phosphorylation genes were significantly downregulated.  We believe that our study makes a significant contribution to the literature because it shows that brood pouch protection in the embryo phase and salinity adaptation in juveniles might be a strategic adaptation to freshwater adaptation in H. heptagonus.Further, we believe that this paper will be of interest to the readership of your journal because the freshwater pipefish adaptation to both fresh and salty water remains unclear. Our study revealed that the brood pouch protects the embryos, and juvenile fish adapt to overcome this osmotic difference.