MAF1 is a global suppressor of RNA polymerase III-dependent transcription, and is conserved from yeast to human. Growing evidence supports the involvement of MAF1 in the immune response of mammals, but its biological functions in fish are unknown. We isolated and characterized Maf1 from the olive flounder Paralichthys olivaceus (PoMaf1). The coding region of PoMaf1 comprised 738 bp encoding a 245-amino-acid protein. The deduced PoMAF1 amino acid sequence shared features with those of MAF1 orthologues from vertebrates. PoMaf1 mRNA was detected in all tissues examined, and the levels were highest in eye and muscle tissue. The PoMaf1 mRNA level increased during early development. In addition, the PoMaf1 transcript level decreased during viral hemorrhagic septicemia virus (VHSV) infection of flounder hirame natural embryo (HINAE) cells. To investigate the role of PoMaf1 in VHSV infection, single-cell-derived PoMaf1 knockout HINAE cells were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system, and cell clones with complete disruption of PoMaf1 were selected. PoMaf1 disruption increased the VHSV glycoprotein (G) mRNA levels during VHSV infection of HINAE cells, implicating PoMAF1 in the immune response to VSHV infection. To our knowledge, this is the first study to characterize fish Maf1, which may play a role in the response to viral infection.

The fish Vitellogenin (Vg) gene has been applied as a biomarker for exposure to estrogenic compounds in the aquatic environment. In this study, we cloned and characterized Vg cDNA from the Korean rose bitterling Rhodeus uyekii (Ru-Vg). The Ru-Vg cDNA encodes a 1424-amino-acid polypeptide that belongs to the VgAo1 family and contains a putative signal peptide, lipovitellin I, phosvitin, and lipovitellin II, but does not contain the vWFD domain or the C-terminal peptide. The deduced Ru-Vg protein has high amino acid identity (73.97%–32.17%) with fish Vg proteins. Pairwise alignment and phylogenetic analysis revealed that Ru-Vg is most closely related to Acheilognathus yamatsutae Vg. Ru-Vg transcripts were detected using quantitative polymerase chain reaction in all tissues tested, with the highest level of expression observed in the ovary. Ru-Vg mRNA was upregulated in R. uyekii hepatopancreas cells in response to treatment with 17β-estradiol (E2) or 17α-ethinylestradiol (EE2). Luciferase reporter expression, driven by the 5'-regulatory region of the Ru-Vg gene spanning from −1020 bp to the start codon was induced by the estrogen receptor and was synergistically activated by treatment with E2 or EE2. These results suggest that R. uyekii and the Ru-Vg gene may be useful as biomarkers for exposure to E2 or EE2.

Thioredoxin is a multifunctional antioxidant enzyme that belongs to the reductase family. In this study, we cloned and characterized thioredoxin 1 cDNA from the Korean rose bitterling Rhodeus uyekii (RuTrx). The full-length RuTrx cDNA consists of 674 bp with a 324 nt open reading frame (ORF) encoding a 107 aa protein. The deduced RuTrx amino acid sequence indicated a characteristic redox active site, ³¹WCGPC³⁵. Pairwise alignment revealed RuTrx amino acid identity (55.1%–83.2%) with orthologs from various species of mammalia, amphibia, fish and bird. Phylogenetic analysis was conducted to determine the evolutionary position of RuTrx. Expression analysis showed that RuTrx transcripts were present in all of the tissues examined, and was high in the hepatopancreas of R. uyekii. During early development, the expression of RuTrx transcripts was increased. Recombinant RuTrx protein (rRuTrx) was tested for its capacity to serve as an antioxidant enzyme using a metal-catalyzed oxidation (MCO) system. The ability of rRuTrx to protect against supercoiled DNA cleavage due to oxidative nicking increased in a dose-dependent manner. In Raw264.7 cells, Dihydroethidium (DHE) staining for ROS production indicated the antioxidant activity of rRuTrx. Together, these findings suggest that RuTrx may play a role in maintaining the redox state balance in Korean rose bitterling R. uyekii.

The olive flounder, Paralichthys olivaceus, also known as the Korean halibut, is an economically important flatfish in East Asian countries. Here, we provided four fully phased genome assemblies of two different olive flounder individuals using high-fidelity long-read sequencing and their parental short-read sequencing data. We obtained 42–44 Gb of ~15-kb and ~Q30 high-fidelity long reads, and their assembly quality values were ~53. We annotated ~30 K genes, ~170-Mb repetitive sequences, and ~3 M 5-methylcytosine positions for each genome assembly, and established a graph-based draft pan-genome of the olive flounder. We identified 5 M single-nucleotide variants and 100 K structural variants with their genotype information, where ~13% of the variants were possibly fixed in the two Korean individuals. Based on our chromosome-level genome assembly, we also explored chromosome evolution in the Pleuronectiformes family, as reported earlier. Our high-quality genomic resources will contribute to future genomic selection for accelerating the breeding process of the olive flounder.

Numerous studies have explored alternative protein sources to fishmeal (FM) to enhance fish diets, yet limited research exists on their effects on maturation. This study assessed the impact of replacing FM with soy protein concentrate (SPC) supplemented with lysine and methionine on growth and gonadal development in olive flounder (Paralichthys olivaceus). Three diets were tested: a control (Con) diet with 60% FM and two diets replacing FM with 25% (LF1) and 50% (LF2) SPC. Fish were fed to apparent satiation twice daily for 12 months. Growth performance and feed intake were not significantly different between groups. However, the gonadosomatic index varied with diet. Muscle composition and amino acid levels were similar across treatments, though n-3 fatty acids were higher in Con. Spermatogonia was increased, and spermatogenesis was impaired in SPC groups. At 12 months, oocytes in Con diets had absorbed yolk globules, whereas this was absent in SPC groups. Growth-related genes in the brain (growth hormones and insulin-like growth factor) were increased with higher SPC, while follicle-stimulating and luteinizing hormones decreased. Estrogen receptor α levels were elevated in SPC groups. Vitellogenin gene expression in gonads was highest in Con, while liver expression peaked in LF2. The expression of digestive enzymes, chymotrypsin2, and trypsin2 was highest in LF2, while lipase genes were lower. In summary, up to 50% FM replacement with SPC, with amino acid supplementation, supported growth performance and muscle composition without adverse effects on growth in olive flounder but influenced gonadal development.