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Elucidating the regulatory mechanisms of ovarian development and oocyte maturation is important for sex-controlled breeding in aquaculture. Among all regulatory pathways, the Notch signaling pathway has been found to play a crucial role in mammalian oocyte development, but its function in teleost has not been reported. In this study, resveratrol, an activator of the Notch signaling pathway, was injected into the ovary of Paralichthys olivaceus in vivo initially, which caused ovary atrophy and partial detachment of internal oocytes. Concurrently, the expression levels of genes related to oocyte development showed significant changes, with which an opposite result could be observed in the in vitro inhibition experiment. To further explore the mechanisms by which Notch signaling are regulated in oocytes, the function of two E3 ubiquitin ligase members (Dtx4) identified from our previous comparative transcriptome analysis was studied. Among them, the expression of dtx4b was significantly higher than that of dtx4a in the ovary. ISH results showed that they were both intensively distributed in oocyte, and they showed different expression patterns during oocyte maturation process. Subsequently, the protein interactions of Dtx4a with Notch1/2- NICD and Dtx4b with Notch2-NICD were verified by fluorescence co-localization and yeast two-hybrid assay. Finally, the negative correlation of dtx4a/b with Notch signaling pathway was verified by dual-luciferase assay and knock down experiment in vitro. These results revealed the regulatory role of dtx4a/b on the Notch signaling pathway for the first time in teleost and provided solid reference for the study of fish gonadal development. • Notch signaling pathway affects ovarian development of P. olivaceus. • dtx4a and dtx4b experienced functional differentiation. • Dtx4a/b regulate Notch signaling pathway by interact with Notch1/2. [ABSTRACT FROM AUTHOR]

Opsins play important roles in the image‐forming visual pathways and numerous biological systems such as the biological clock and circadian rhythm. However, the nonvisual opsins involved in nonimage forming process are not clear to date. The aim of this study was to characterize nonvisual opsins in Paralichthys olivaceus. A total of 24 nonvisual opsin genes were identified. Expressions of these genes in eye, brain, heart, testis, and fin were investigated by quantitative real‐time polymerase chain reaction (qRT‐PCR). Testis contained a surprisingly large number of nonvisual opsins including Opn4m2, Tmt2a, Tmt3b, Opn3, RRH, Opn7a, and Opn7b. Syntenic and phylogenetic analyses confirmed that the RGRa and RGRb originated from the teleost‐specific genome duplication (TSGD). qRT‐PCR results demonstrated high RGRa and RGRb expression in the eye, while the expression levels in the brain, heart, testis, and fin were relatively weak. In situ hybridization results presented here revealed the presence of both RGRa and RGRb mRNA‐positive signals in the ganglion cell layer but absence in the intracellular compartment of retinal pigment epithelium (RPE) and Müller glial cells. Therefore, we hypothesized that RGRa and RGRb had undergone subfunctionalization in P. olivaceus after TSGD. In conclusion, this study provides novel insights into the evolutionary fates of the RGR genes, still, further studies need to be done to explore the mechanism about the lack of RGR genes' expression in RPE. Research Highlights: Non‐visual opsins involve in non‐image forming are studied in flounder. Testis contained large number of non‐visual opsins. RGRa/b originated from TSGD have functionally diversified. The results provided novel insights into the evolution RGRs. [ABSTRACT FROM AUTHOR]

To study the effect of Edwardsiella tarda infection on miRNAs expression profile in Japanese flounder, fish were injected intraperitoneally with E. tarda. The miRNAs involved in regulating immune responses were analyzed by high-throughput sequencing. A total of 164 mature miRNAs were identified, of which 17 miRNAs were differentially expressed (DE miRNAs) after E. tarda infection, indicating that they were immune-related miRNAs. To further examine the relationship between the miRNAs and their predicted target mRNAs, a total of 22 predicted target mRNAs, mainly related to endocytic signaling pathway, NF- κ B signaling pathway, and p53 signaling pathway, were detected with miRNA mimics in HEK-293T cells by dual-luciferase reporter experiments. Finally, we confirmed that insulin receptor substrate-2 (IRS2a and IRS2b) were regulated by miR-7a. And the target sites of the 3' untranslated region (UTR) of IRS2a and IRS2b were verified by dual-luciferase reporter experiments. Furthermore, we found that the E. tarda and LPS significantly increased host miR-7a expression. In vivo and in vitro studies revealed that IRS2-mediated PI3K/AKT/GSK3β signaling pathway was suppressed. Taken together, these results implied that miR-7a might be a key regulator of PI3K/AKT/GSK3β signaling pathway via suppressing the IRS2a and IRS2b genes. • Totals of 164 mature miRNAs were identified from Paralichthys olivaceus liver. • Seventeen miRNAs were differentially expressed post Edwardsiella tarda infection. • IRS2a and IRS2b were target genes of miR-7a. • miR-7a was maybe a new negative regulator of IRS2-mediated PI3K/AKT/GSK3β pathway. [ABSTRACT FROM AUTHOR]

A newly isolated Japanese flounder (Paralichthys olivaceus) β-actin promoter and its derivative compact construct Poβ-actinΔ−1080/−801Δ−500/−201 have recently been demonstrated to promote ectopic gene expression in cell lines. Different Poβ-actin promoter deletion mutants were constructed and functionally characterized. Mutational analyses by dual-luciferase detected that three regulatory elements, including one enhancer (−1399/−1081) and two silencers (−1080/−801, −500/−201) in the first intron. The sequence located at −1399/−1081 was determined to significantly affect promoter activity. Additionally, the first exon (−1489/−1400) could also remarkably promote the β-actin promoter activity. In the following transduction application, we removed the two silencers and generated a compact reconstruct promoter/enhancer (Poβ-actinΔ−1080/−801Δ−500/−201), which exhibited relatively stronger promoter activity compared with Poβ-actin. Furthermore, the green fluorescent protein (GFP) transgenic stable flounder cell line was obtained by the reconstructed Poβ-actinΔ−1080/−801Δ−500/−201 promoter. Our study provided the potential application of Japanese flounder β-actin, particularly Poβ-actinΔ−1080/−801Δ−500/−201, in ectopic gene expression in the future. [ABSTRACT FROM AUTHOR]

Cystatin B is an intracellular inhibitor that regulates the activities of cysteine proteases. In this study, cystatin B in Japanese flounder ( Paralichthys olivaceus ) was characterized and its immune function was analyzed. This gene had a high similarity with the sequence of cystatin B in other fish species, and the derived peptide shared typical features of cystatin proteins including the QXVXG motif. The results of quantitative real-time PCR showed that cystatin B mRNA was constitutively expressed in all examined tissues, with the highest level in gill. The stimulations of lipopolysaccharide, peptidoglycan and polyinosinic-polycytidylic acid effectively increased the expression level of cystatin B mRNA. Functional analysis implied that the recombinant P. olivaceus cystatin B purified from Escherichia coli had cysteine protease inhibitory activity and could inhibit bacterial growth by binding to bacteria. Furthermore, we found that P. olivaceus cystatin B had no effects on the expression of inflammatory factors cytokines tumor necrosis factor α, interleukin 10, interleukin 1β and interferon γ. These results indicate that cystatin B of P. olivaceus is potentially involved in immune responses against invading microbial pathogens, and provide a better understanding of the immune mechanisms of cystatins in teleosts. [ABSTRACT FROM AUTHOR]

Marine organisms are under a frequent threat from various pathogens. Edwardsiella tarda is one of the major fish pathogens infecting both cultured and wild fish species. It can also infect a variety of other vertebrates, including amphibians, reptiles, and mammals, and bacteremia caused by E. tarda can be fatal in humans. The kidney is the largest lymphoid organ in fish, and generating kidney transcriptomic information under different stresses is crucial for understanding molecular mechanisms underlying the immune responses in the kidneys. In this study, we performed transcriptome-wide gene expression profiling of the Japanese flounder ( Paralichthys olivaceus ) challenged by 8 and 48 h of E. tarda infection. An average of 40 million clean reads per library was obtained, and approximately 81.6% of these reads were successfully mapped to the reference genome. In addition, 1319 and 4439 differentially expressed genes (DEGs) were found at 8 and 48 h post-injection, respectively. Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to search immune-related DEGs. A protein–protein interaction network was constructed to ascertain the relationship between interacting immune genes during pathogen-induced stress. Based on the KEGG and protein association networks analysis, 24 hub genes were discovered and validated by qRT-PCR. To our knowledge, this study is the first to represent the kidney transcriptome analysis based on protein–protein interaction networks in fish. Our results provide valuable gene resources for further research on kidney immune response in fish, which can significantly improve our understanding of the molecular mechanisms underlying the immune response to E. tarda in humans and other vertebrates. [ABSTRACT FROM AUTHOR]

Abstract Krüppel-like factor 4 (Klf4) is a zinc finger protein transcription factor of the KLF family and plays an important role in various important biological process, including cell proliferation, differentiation, embryonic development, and cancer formation. In this study, the full length Klf4 cDNA sequence of Japanese flounder (Paralichthys olivaceus) was analyzed. Phylogenetic, gene structure, and synteny analysis revealed that PoKlf4 was conserved to that of teleost. Transcript of PoKlf4 showed that its expression was high during early embryonic development but reduced when gastrulation began, thus indicating its possible role in embryonic development. Tissue expression pattern showed that PoKlf4 was expressed higher in ovaries than in testes. ISH revealed that PoKlf4 was also expressed in spermatogonium, oocytes, and oogonia and in high levels in epithelial and lamellar epithelial cells of gills. Functional characterization of PoKlf4 gene promoter provided valuable information in understanding the molecular regulatory mechanisms of PoKlf4 gene. PoKlf4 could up-regulate Nanog , a gene that prevents ES cells from differentiating and being up-regulated by Pou5f3 and Stat3 genes that could maintain ES cell self-renewal. These results suggested that PoKlf4 maintains the pluripotency of ES cells. This study provides valuable information in exploring the regulatory network of PoKlf4 and lays the foundation for further research on the function of this gene. Highlights • The cDNA of Klf4 gene was isolated and characterized from Japanese flounder. • The expression profiles of Klf4 were analyzed by qRT-PCR and ISH. • PoKlf4 could up-regulate Nanog , and PoKlf4 was regulated by Pou5f3 and Stat3. [ABSTRACT FROM AUTHOR]

The COMMD (Copper Metabolism gene MuRR1 Domain) gene family, containing the COMM domain, is highly conserved in diverse lineages and participate in many cellular and biological processes, such as iron transport, regulation of NF-κB activity, and cell proliferation. However, the characteristics and functions of COMMD family are never described in teleost species. Here, we identified all ten COMMD family members in Japanese flounder (Paralichthys olivaceus) based on the genomic and transcriptomic data. COMMD members displayed diverse expression patterns in eleven investigated tissues of Japanese flounder. The regulation of COMMDs in Japanese flounder exposed to Edwardsiella tarda challenge in three immune-related organs, liver, intestine and gill suggested their possible role in innate immune response. Moreover, the expression levels of COMMDs were also significantly up-regulated after LPS stimulation in gill cell line. We further confirmed that PoCOMMD1 could suppress the activity of NF-κB through COMM domain. Taken together, our study elaborates the characteristics and functions of COMMDs in innate immune response of a teleost species. This adds more information for understanding the evolutionary dynamics and immune-related functions of COMMD family in teleost. • A full set of 10 COMMD genes were identified and characterized in Paralichthys olivaceus genome. • The COMMD genes were regulated in a spatio-temporal specific pattern after bacterial stimulation. • The COMMD family genes were involved in innate immune response in Paralichthys olivaceus • COMMD1 could inhibit the activity of NF-κB depends on COMM domain. [ABSTRACT FROM AUTHOR]