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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 a cold-water fish widely cultured in Northeast Asia. With global warming and increased breeding density, Japanese flounder aquaculture is threatened by heat stress. Therefore, understanding the molecular mechanisms underlying heat stress is important. This study applied two laboratory heat stress conditions, gradual temperature rise (GTR) and abrupt temperature rise (ATR) to imitate the stresses faced by Japanese flounder. The histological damage of spleen and the underlying molecular mechanisms were revealed. Histological observation showed that the size and number of melanoma macrophage centers (MMCs) changed under the applied heat stresses; however, inflammatory infiltration and erythrocytes specifically appeared only under the ATR stress. Transcriptome analysis identified 1073 and 985 differentially expressed genes (DEGs) in the GTR vs. the C and the ATR vs. the C groups, respectively. Functional enrichment analysis illustrated GTR and ATR stresses affected similar categories while simultaneously influencing diverse functions. The "Protein processing in endoplasmic reticulum (ER)" was the most enriched pathway in two kinds of heat stress. The "Apoptosis" pathway was GTR-specific while the"DNA replication" was ATR-specific. In addition, the comparison between GTR and ATR stresses in "Protein processing in ER" pathway were further described. The results illustrated both acute and chronic heat stress could increase misfolded proteins, disrupt the ER function, and cause histological damage. Misfolded proteins in the GTR group were removed by apoptosis, whereas those in the ATR group were cleared via DNA-related pathways. Protein-Protein Interaction (PPI) analysis identified 20 hub genes, eight of which were shared between the two groups (hsp90aa1.1 , hspa5 , hsp90b1 , hsp90ab1 , hspa9 , hsp70 , hspe1 and hspd1); three (hspa4b , fn1a and bmp4) were GTR-specific, whereas nine genes (chek1 , pcna , aurkb , rpal , cdc6 , rrm1 , mcm5 , pold1 and mcm3) were ATR-specific. The current study sheds light on the molecular mechanisms underlying the heat stress response in Japanese flounder. • Heat stress damaged the histological structure of spleen. • More serious damage was observed in ATR group in compared to GTR group. • The functional enrichment analysis of DEGs in GTR and ATR stresses showed commonality and specificity. • Heat stress could increase misfolded proteins and disrupt the ER function, then caused the spleen histological damage. • A total of twenty hub genes were identified as the key genes of Japanese flounder in response to heat stress. [ABSTRACT FROM AUTHOR]

• A total of 14 mapk genes were identified and characterized in Japanese flounder genome. • The mapk genes were evolving-constrained and function conserved. • The mapk genes play an important role after biotic and abiotic stresses. Mitogen-activated protein kinase (MAPK) are a series of serine/threonine protein kinases showing evolutionary conservation, which can be activated by many stimulus signals and then transfer them from cell membrane to nucleus. MAPKs regulate a variety of biological processes, such as apoptosis, hormone signaling and immune response. In this study, 14 putative mapk genes in Japanese flounder were identified, and their basic physical and chemical properties were characterized. Phylogenetic analysis showed that mapk genes were divided into three main subfamilies, including ERK, JNK and the p38 MAPK. Selection pressure analysis revealed they were evolutionarily-constrained and undergone strong purifying selection. Gene structure and conserved protein motif comparison suggested high levels of conservation in members of mapk gene family. The expression patterns were further investigated in each embryonic and larval development stages and different tissues. In addition, RNA-seq analyses after bacteria and temperature stresses suggested mapk genes had different expression patterns. Three mapk genes showed significant differences in response to E. tarda challenge and five were induced significantly after temperature stress, indicating their potential functions. This systematic analysis provided valuable information for further understanding of the regulation mechanism of mapk gene family under different stresses in Japanese flounder. [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]