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As an economically important flatfish in Asia, Japanese flounder is threatened by continuously rising temperatures due to global warming. To understand the molecular responses of this species to temperature stress, adult Japanese flounder individuals were treated with two kinds of heat stress—a gradual temperature rise (GTR) and an abrupt temperature rise (ATR)—in aquaria under experimental conditions. Changes in histopathology, programmed cell death levels and the oxidative stress status of gills were investigated. Histopathology showed that the damage caused by ATR stress was more serious. TUNEL signals confirmed this result, showing more programmed cell death in the ATR group. In addition, reactive oxygen species (ROS) levels and the 8-O-hDG contents of both the GTR and ATR groups increased significantly, and the total superoxide dismutase (T-SOD) activities and total antioxidant capacity (T-AOC) levels decreased in the two stressed groups, which showed damage to antioxidant systems. Meanwhile, RNA-seq was utilized to illustrate the molecular mechanisms underyling gill damage. Compared to the control group of 18 °C, 507 differentially expressed genes (DEGs) were screened in the GTR group; 341 were up-regulated and 166 were down-regulated, and pathway enrichment analysis indicated that they were involved in regulation and adaptation, including chaperone and folding catalyst pathways, the mitogen-activated protein kinase signaling (MAPK) pathway and DNA replication protein pathways. After ATR stress, 1070 DEGs were identified, 627 were up-regulated and 423 were down-regulated, and most DEGs were involved in chaperone and folding catalyst and DNA-related pathways, such as DNA replication proteins and nucleotide excision repair. The annotation of DEGs showed the great importance of heat shock proteins (HSPs) in protecting Japanese flounder from heat stress injury; 12 hsp genes were found after GTR, while 5 hsp genes were found after ATR. In summary, our study records gill dysfunction after heat stress, with different response patterns observed in the two experimental designs; chaperones were activated to defend heat stress after GTR, while replication was almost abandoned due to the severe damage consequent on ATR stress. [ABSTRACT FROM AUTHOR]

Japanese flounder (Paralichthys olivaceus) is an economically important flatfish endemic to Asia that faces natural challenges, such as heat shock and bacterial infection. Here, we investigated whether heat shock protein 20 (hsp20) in Japanese flounder played a role in immune reactions against Edwardsiella tarda. Hsp20 is ubiquitously produced by cells in the stress response; it not only functions as a chaperone and housekeeping protein but also participates in the immune response during infection. In this study, we identified and annotated 13 hsp20 genes using bioinformatic methods in Japanese flounder. Phylogenetic and selection pressure analysis showed that the hsp20 genes were evolutionarily-constrained and their function was conserved. Hsp20 genes are also involved in the embryogenesis and act as the sentinel role in an unchallenged organism. Moreover, the regulated expression patterns of hsp20 genes after E. tarda infection were examined for the first time in Japanese flounder. Eleven out of 13 genes were differentially expressed after bacterial challenge, which indicated that they may be involved in disease response genes or as disease defence against pathogens. These results provide valuable information for clarifying the evolutionary relationship of the hsp20 genes, and early insights into the immune functions of hsp20 genes in Japanese flounder. • A full set of 13 hsp20s were identified and characterized in Japanese flounder genome. • The hsp20 genes were evolving-constrained and function conserved by phylogenetic and selection pressure analysis. • Most hsp20 genes were regulated in a time-specific, tissue-specific pattern after bacterial infection. [ABSTRACT FROM AUTHOR]