Flounder Paralichthys Olivaceus Research Articles

Paralichthys olivaceus GSDME-mediated pyroptosis is regulated by multiple caspases in different manners

Pyroptosis is a type of programmed cell death mediated by gasdermin (GSDM). GSDM is activated by caspase (CASP), which cleaves GSDM to release the N-terminal (NT) fragment that forms channels in the plasma membrane and leads to cell death. To date, research on pyroptosis in teleost is limited. In this study, we examined the activation and regulation mechanism of pyroptosis in flounder Paralichthys olivaceus. P. olivaceus gasdermin E (PoGSDME) was found to be cleaved by six P. olivaceus caspases (PoCASP1/3a/3b/7/8a/8b). PoCASP1/3a/3b/7 cleaved primarily at 245FEAD248, which generated an NT fragment (NT248) that induced robust pyroptosis. PoCASP8a/8b cleaved both the full length PoGSDME and NT248 at 202IEKD205, thus destroying the biological activity of PoGSDME and NT248. Nine residues crucial for PoGSDME function were identified, of which, F2, L19, and G85 were essential to plasma membrane translocation. During bacterial infection, PoGSDME and PoCASP1 expressions were significantly upregulated in flounder tissues, and PoGSDME, as well as PoCASP1, activation occurred in flounder cells accompanied with the processing cleavage of IL-1β and IL-18. Together these results revealed both the activation and the inhibition mechanisms of GSDME-mediated pyroptosis in flounder, and added new insights into the regulation of pyroptosis in fish.

CRADD and cIAP1 antagonistically regulate caspase-9-mediated apoptosis in teleost

Caspase 9 (CASP9) is a well-known initiator caspase of intrinsic apoptosis. In humans, cIAP1 binds and induces degradation of the activated form of CASP9, but not pro-CASP9. In fish, the activity and regulation of CASP9 remain unknown. In this work, using flounder Paralichthys olivaceus as a representative species, we examined the regulatory mechanism of CASP9 in teleost. P. olivaceus CASP9 (PoCASP9) induced robust apoptosis, which was inhibited by P. olivaceus cIAP1 (PocIAP1). Unlike human cIAP1, PocIAP1 bound both pro- and active PoCASP9 and induced their degradation via the RING domain-involved proteasome pathway. In humans, the adaptor molecule CRADD cannot interact with CASP9. In contrast, P. olivaceus CRADD (PoCRADD) bound both pro- and active PoCASP9 via CARD-CARD interaction and enhanced apoptosis by promoting the cellular levels of pro- and active PoCASP9. Furthermore, PoCRADD abrogated the inhibition of PoCASP9 by PocIAP1 by preventing PocIAP1-PoCASP9 interaction. Together these results reveal a CASP9 regulation mechanism in teleost that differs from that in humans and demonstrate that teleost CASP9 is tightly and directly controlled by both negative and positive regulators that exert a regulation effect both before and after CASP9 activation. These findings advance our understanding of the regulation of CASP9-mediated apoptosis in vertebrates.

The miR-200 Family Targeting amh Affects the Gonadal Development of Japanese Flounder

Four members of the miR-200 family in Japanese flounder (Paralichthys olivaceus) have sex-biased expression patterns, but their target genes and how they work in the development of the gonads are rarely known. Anti-Müllerian hormone (AMH) can inhibit the development of Muller’s duct in female mammals and regulate the formation of gametes after sexual maturity. There is no Muller’s duct in teleosts, but the amh gene still exists. Knockout of amh results in sex reversal from male to female. Therefore, it is essential to explore the relationship between the miR-200 family and amh to clarify what role miR-200 plays in the development of the gonads. In Japanese flounder, the two binding sites for the miR-200 family in the 3′UTR of amh were found through bioinformatic prediction. Double luciferase and green fluorescent protein reporter experiments demonstrated amh to be directly targeted by miR-200a and miR-200b. Moreover, miR-200a and miR-200b reduced the expression of amh through site 1 rather than site 2. To explore the regulatory role of miR-200a in gonadal development, we further overexpressed miR-200a in the primary Sertoli cells of the testis. With the overexpression of miR-200a, the expression of amh decreased, while the expression of the other two male sex-related genes, dmrt1 (doublesex and mab-3 related transcription factor 1) and gsdf (diagonal soma driven factor), increased significantly. This result indicates that the miR-200 family regulates the gonadal differentiation and development by targeting amh in Japanese flounder.

Otolith development and elemental incorporation in response to seawater acidification in the flounder Paralichthys olivaceus at early life stages

Ocean acidification can influence the formation, development and functions of calcified structures in marine organisms, such as otoliths, which are mainly composed of calcium carbonate (CaCO3) and function in orientation, balance, sensory perception and locomotion in fish. This study investigated the impacts of seawater acidification (pH 8.10, 7.70 and 7.30, roughly corresponding to the ocean acidification under RCP 8.5 scenario predicted by the IPCC) on somatic growth, otolith (aragonite) morphology and microchemistry in the flounder Paralichthys olivaceus at early life stages (ELSs, exposed to acidified seawater via pCO2 from embryonic to juvenile stages for 52 days). The results demonstrated that seawater acidification promoted otolith growth (mass and size) but did not change their geometric outlines. Seawater acidification did not alter the somatic growth or otolith elemental incorporation (Sr, Ba and Mg) in the flounder. Seawater acidification increased the occurrence of abnormally developed calcitic otoliths (calcite) which considerably differed from the aragonitic otoliths in surface and crystal structures. Additionally, elemental incorporation (Sr:Ca and Ba:Ca) appeared to be higher in aragonitic otoliths than in calcitic otoliths, which was likely related to their unique manners of formation. Our results agreed with the broad literature, in that seawater acidification showed species-specific influences (positive or no effect) on otolith size but did not affect somatic growth, otolith shape or elemental incorporation of fish at ELSs. These findings provide knowledge for evaluating the ecological effects of ocean acidification on the recruitment and population dynamics of fish in the wild.

Two bicistronic DNA vaccines against Vibrio anguillarum and the immune effects on flounder Paralichthys olivaceus.

Chemokines are cytokines that can promote the activation and migration of immune cells, and increase the recognition of antigen by antigen-presenting cells (APC). Previous studies showed that a DNA vaccine can induce humoral and cellular immune responses of flounder after immunization. To explore the improvement of chemokines on the efficiency of OmpK vaccine, two bicistronic DNA candidate vaccines were constructed and the immune responses they induced in the flounder were investigated by reverse transcription polymerase chain reaction (RT-PCR), indirect immunofluorescent assay (IFA), H&E staining, flow cytometry (FCM), and quantificational real-time polymerase chain reaction (qRT-PCR). pBudCE4.1 plasmid as an expression vector, bicistronic DNA vaccines encoding OmpK gene and CC-motif ligand 4 gene (p-OmpK-CCL4), or Ompk gene and CC-motif ligand 19 gene (p-OmpK-CCL19) were successfully constructed. The results showed that two bicistronic DNA vaccines expressed Ompk protein of Vibrio anguillarum and CCL4/CCL19 proteins of flounder both in vitro and in vivo. After immunization, a large number of leucocytes in muscle were recruited at the injection site in treatment groups. The constructed vaccines induced significant increases in CD4-1+ and CD4-2+ T lymphocytes, and sIgM+ B lymphocytes in peripheral blood, spleen, and head kidney. The percentage of T lymphocytes peaked on the 14th post-vaccination day whereas that of B lymphocytes peaked in the 6th post-vaccination week. Moreover, the expression profiles of 10 immune-related genes increased in muscles around the injection site, spleen, and head kidney. After the challenge, p-OmpK-CCL4 and p-OmpK-CCL19 conferred a relative percentage survival (RPS) of 74.1% and 63.3%, respectively, higher than p-OmpK alone (40.8%). In conclusion, both CCL4 and CCL19 can improve the protection of p-OmpK via evoking local immune response and then humoral and cellular immunity. CCL4 and CCL19 will be potential molecular adjuvants for use in DNA vaccines.Electronic supplementary materialSupplementary material (Supplementary Material) is available in the online version of this article at 10.1007/s00343-021-1092-z.

Brown seaweed ( Sargassum fulvellum ) inclusion in diets with fishmeal partially replaced with soy protein concentrate for Japanese flounder ( Paralichthys olivaceus ) juveniles

Brown seaweed ( <i>Sargassum fulvellum</i> ) inclusion in diets with fishmeal partially replaced with soy protein concentrate for Japanese flounder ( <i>Paralichthys olivaceus</i> ) juveniles

Two CD9 tetraspanin family members of Japanese flounder (Paralichthys olivaceus): characterization and comparative analysis of the anti-infectious immune function

CD9 is a glycoprotein of the transmembrane 4 superfamily that is involved in various cellular processes. Studies related to the immune functions and activities of CD9 in teleost fish are limited. In this study, we characterized two CD9 homologs, PoCD9.1 and PoCD9.3, from Japanese flounder (Paralichthys olivaceus). Sequence analysis showed that PoCD9.1 and PoCD9.3 possess characteristic transmembrane 4 superfamily (TM4SF) structures. PoCD9.1 shares 70.61% sequence identity with PoCD9.3. The expression of PoCD9.1 and PoCD9.3 in the three main immune tissues was significantly induced in a time-dependent manner by extracellular and intracellular pathogen infection, which indicates that the two CD9 homologs play an important role in the response to pathogenic infection. Following infection with the extracellular pathogen Vibrio anguillarum, the expression profiles of both PoCD9.1 and PoCD9.3 were similar. After infection with the intracellular pathogen Edwardsiella piscicida, the expression levels of PoCD9.1 and PoCD9.3 were different at different stages of infection, especially in the spleen. The spleen was the most important tissue for the PoCD9.1 and PoCD9.3 responses to pathogen infection among the three examined immune tissues. Knockdown of PoCD9.1 and PoCD9.3 attenuated the ability of host cells to eliminate pathogenic bacteria, and PoCD9.1 knockdown was more lethal than PoCD9.3 knockdown for host cells with E. piscicida infection. Overexpression of PoCD9.1 and PoCD9.3 promoted host or host cell defence against E. piscicida infection. These findings suggest that PoCD9.1 and PoCD9.3 serve as immune-related factors, play an important role in the immune defence system of Japanese flounder, and display different functions in response to different pathogens at different stages of infection.

Targeted mutagenesis in the olive flounder (Paralichthys olivaceus) using the CRISPR/Cas9 system with electroporation

As a new breeding technology, genome editing becomes a powerful tool owing to its high efficiency of gene targeting. In CRISPR/Cas9 system, how to efficiently transfer gRNA and Cas9 mRNA into embryos is an important step. Though microinjection is the most common method for operating on fish embryos, it is not easy to inject the RNA into pelagic and telolecithal eggs with hard egg chorion, such as the olive flounder (Paralichthys olivaceus) eggs. Therefore, an efficient and simple technology is urgently needed for this kind of study. In the present study, we used the electroporation method to introduce foreign gene into the flounder eggs. The results showed that the proper electroporation condition was 3 pulses for 1 millisecond (ms), 50 ms interval, at 25 V with high survival rate. Under this condition, the effect of CRISPR/Cas9 system on genome editing by using two different genes, myomaker and gonadal soma derived factor (gsdf) was investigated. Around 12% and 7% of the electroporated embryos for myomaker and gsdf hatched, respectively. The mutation sites including insert and deletion mutations at the candidate sites were visible for both targeted genes in the hatched larvae. The checked frame-shift and start codon deletion mutations would lead to complete destruction of these genes’ structure. Above results implied that CRISPR/Cas9 system could work well in marine fish with pelagic eggs by using electroporation, and genome editing could be achieved on a large scale which may be useful for study of gene function in marine fish.

Expression of circadian rhythm-related hormones in juvenile olive flounders (Paralichthys olivaceus) following exposure to total residual oxidant

ABSTRACT Some substances used in aquaculture may be toxic to fish depending on their concentration. Here, we investigated the changes in circadian rhythm in olive flounders (Paralichthys olivaceus) exposed to total residual oxidant (TRO), a substance produced by the reaction of O3 with Br− ions, at two different concentrations (20 and 40 μg/L) for different periods (2, 6, 10, 14, 18, 22, 30, and 42 h) to identify the optimum concentration of TRO for aquaculture. We analysed mRNA expression and the activity of circadian rhythm-related hormones (period 2 (Per2), cryptochrome 1 (Cry1), melatonin receptor 1 (MT-R1), and melatonin) in the diencephalon and plasma. The levels of circadian rhythm-related hormones in fish exposed to 40 μg/L TRO were significantly lower than those in the other groups. However, there were no significant changes in circadian rhythm-related hormone levels in fish exposed to 20 μg/L TRO. These results indicate that 20 μg/L TRO does not significantly affect the circadian rhythms in the olive flounder and that it is suitable for sterilisation purposes in aquaculture.

Pharmacodynamics of Ceftiofur Selected by Genomic and Proteomic Approaches of Streptococcus parauberis Isolated from the Flounder, Paralichthys olivaceus.

We employed an integrative strategy to present subtractive and comparative metabolic and genomic-based findings of therapeutic targets against Streptococcus parauberis. For the first time, we not only identified potential targets based on genomic and proteomic database analyses but also recommend a new antimicrobial drug for the treatment of olive flounder (Paralichthys olivaceus) infected with S. parauberis. To do that, 102 total annotated metabolic pathways of this bacterial strain were extracted from computational comparative metabolic and genomic databases. Six druggable proteins were identified from these metabolic pathways from the DrugBank database with their respective genes as mtnN, penA, pbp2, murB, murA, coaA, and fni out of 112 essential nonhomologous proteins. Among these hits, 26 transmembrane proteins and 77 cytoplasmic proteins were extracted as potential vaccines and drug targets, respectively. From the FDA DrugBank, ceftiofur was selected to prevent antibiotic resistance as it inhibited our selected identified target. Florfenicol is used for treatment of S. parauberis infection in flounder and was chosen as a comparator drug. All tested strains of fish isolates with S. parauberis were susceptible to ceftiofur and florfenicol with minimum inhibitory concentrations (MIC) of 0.0039–1 μg/mL and 0.5–8 μg/mL, IC50 of 0.001–0.5 μg/mL and 0.7–2.7 μg/mL, and minimum biofilm eradication concentrations (MBEC) of 2–256 μg/mL and 4–64 μg/mL, respectively. Similar susceptibility profiles for ceftiofur and florfenicol were found, with ceftiofur observed as an effective and potent antimicrobial drug against both planktonic and biofilm-forming strains of the fish pathogen Streptococcus parauberis, and it can be applied in the aquaculture industry. Thus, our predictive approach not only showed novel therapeutic agents but also indicated that marketed drugs should also be tested for efficacy against newly identified targets of this important fish pathogen.

Draft Genome Sequences of Two Edwardsiella piscicida Strains, JF1307 and JF1411, Isolated from Diseased Olive Flounder (Paralichthys olivaceus) Cultured in Japan.

Edwardsiella piscicida strains JF1307 and JF1411 were isolated from cultured olive flounder that were diagnosed as being infected with edwardsiellosis. The draft genome sequences of the two isolates comprise 3,882,000 bp and 3,827,424 bp with G+C contents of 59.5% and 59.6%, respectively.

Generation, characterization and application of monoclonal antibodies against matrix protein of hirame novirhabdovirus (HIRRV) in flounder.

Hirame novirhabdovirus (HIRRV) causes severe disease in fish cultures, resulting in great economic loss in Asia and Europe. In this study, the matrix protein (M) of HIRRV was recombinantly expressed as the immunogen to produce monoclonal antibodies (MAbs) using hybridoma cell fusion technology, and 3 MAbs were produced and characterized by indirect ELISA, Western blotting and immunofluorescence assay (IFA). Western blotting and mass spectrometric analysis showed that the MAbs could specifically react with the nature M protein of HIRRV. The MAbs were employed to detect virions in HIRRV-infected epithelioma papulosum cyprini (EPC) cells and flounder Paralichthys olivaceus by IFA and immunohistochemistry (IHC). In the virus-infected EPC cells, the virions were mainly located in the cytoplasm, whereas in flounder, HIRRV was present in all 10 tested tissues, and the positive signals in spleen, head-kidney and heart were higher than in other tissues, consistent with the results obtained by RT-PCR. Moreover, strong positive signals were observed in the endothelial cells of blood vessels, but only the leukocytes were infected by HIRRV in the whole blood cells. These results indicate that the high susceptibility to HIRRV of leukocytes and endothelial cells may facilitate the spread of HIRRV and finally cause systemic infection in flounder. This study provides a foundation for further studies on rapid diagnosis of HIRRV and its infection mechanisms.

Compensatory Growth of the Juvenile Brown Flounder Paralichthysolivaceus Following Being Kept at High Temperature for a Single Phase of Different Length

Compensatory Growth of the Juvenile Brown Flounder Paralichthysolivaceus Following Being Kept at High Temperature for a Single Phase of Different Length

Identification of transposable elements fused in the exonic region of the olive flounder genome.

Transposable elements (TEs) are mobile genetic sequences that comprise a large portion of vertebrate genomes. The olive flounder (Paralichthys olivaceus) is a valuable marine resource in East Asia. The scope of most genomic studies on the olive flounder is limited to its immunology as their focus is the prevention of mass mortality of this species. Thus, for a broader understanding of the species, its genomic information is consistently in demand. Transcripts sequences were acquired from transcriptome analysis using gill tissues of 12 olive flounders. Distribution of TEs inserted in exonic region of the olive flounder genome was analyzed using RepeatMasker ( http://www.repeatmasker.org/ ). We found 1140 TEs in the exonic region of the genome and long interspersed nuclear elements (LINEs) and long terminal repeats (LTRs) insertions occurred with forward orientation preferences. Transposons belonging to the hAt, Gypsy, and LINE 1 (L1) subfamilies were the most abundant DNA transposons, LTRs, and long interspersed elements (LINEs), respectively. Finally, we carried out a gene ontology analysis to determine the function of TE-fused genes. These results provide some genomic information about TEs that is useful for future research on changes in properties and functions of genes by TEs in the olive flounder genome.

Evaluation of the inflammatory response to Kudoa septempunctata genotype ST3 isolated from olive flounder (Paralichthys olivaceus) in Caco-2 cells.

Kudoa septempunctata (Myxosporea, Multivalvulida) is a parasite of the trunk muscle of cultured olive flounder (Paralichthys olivaceus). We investigated whether K. septempunctata genotype ST3 spores induce cell damage and the secretion of inflammatory mediators in Caco-2 cells, which exhibit characteristics similar to human intestinal epithelial cells. Purified K. septempunctata spores were heated at 95 °C for 5 min. Lactate dehydrogenase (LDH) release was measured to determine the efficacy of denaturation. Naïve and heated spores, lipopolysaccharide (positive control) and vehicle (negative control) were added to Caco-2 cells. Cells were subjected to the cytotoxic LDH assay and western blot analysis to examine the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. Supernatants were collected to measure nitric oxide (NO) and prostaglandin E2 (PGE2). Most spores were denaturated by heating, and the spore morphology was found to be wrinkled with shell valves and polar capsules. In addition, cytotoxicity and inflammatory mediators, such as NO, PGE2, iNOS, and COX-2, remained unchanged in Caco-2 cells following exposure to naïve and heated spores compared with the positive controls. Collectively, the findings of this study imply that spores of K. septempunctata genotype ST3 do not cause inflammation in Caco-2 cells.

Localization and dynamic expression of a 27.8 kDa receptor protein for lymphocystis disease virus infection in sea bass (Lateolabrax japonicus) tissues

Lymphocystis disease virus (LCDV) infects target cells by attaching to a 27.8 kDa receptor (27.8R) protein in flounder Paralichthys olivaceus, and anti-27.8R monoclonal antibodies (MAbs) have been developed. However, the 27.8R existence in tissues of sea bass (Lateolabrax japonicus) and its role in LCDV infection have remained unclear. In this study, the results of western blotting demonstrated that the same 27.8R was shared by flounder and sea bass. LCDV-free sea bass individuals were intramuscularly injected with LCDV, and viral copies were detected in tissues from 3 h post infection and showed a time-dependent increase during 9 days infection. Distribution and synthesis of 27.8R in sea bass tissues were investigated by using anti-27.8R MAbs as probes. It was found that 27.8R was distributed in all the tested tissues. The levels of 27.8R protein were highest in gill and skin, then a bit lowly in stomach, head kidney and heart, followed by spleen, intestine, blood cells, gonad and liver, and least in kidney and brain in healthy sea bass. Upon LCDV infection, 27.8R synthesis was up-regulated in each tissue, and higher in the tissues with higher LCDV copies. The 27.8R and LCDV were detected in some peripheral blood leukocytes but not in red blood cells. These results suggested that 27.8R was widely distributed in sea bass tissues, and it served as a receptor and correlated with tissue tropism of LCDV infection. Furthermore, leukocytes had the potential of being a LCDV carrier and were responsible for a systemic infection of LCDV in sea bass.

Identification of candidate piRNAs in the gonads of Paralichthys olivaceus (Japanese flounder).

Piwi-interacting RNA (piRNA) plays an important role in the gonadal development and maintenance of Teleostei. In this study, piRNA libraries derived from the adult gonads of Japanese flounder (Paralichthys olivaceus) were generated using next-generation sequencing technology. Using zebrafish piRNAs as a reference, 5 865 unique candidate piRNAs were identified; 289 candidate piRNA clusters (PRCs) were generated from the above piRNAs. Among the isolated candidate PRCs, a total of 38 ovary-specific, 45 ovary-bias, 24 testis-specific, and 131 testis-bias PRCs were found. The relative expression levels of seven PRCs were validated through quantitative reverse transcription-polymerase chain reaction. The results of this study will help facilitate exploration of the development and maintenance of the phenotypic sex mechanism in P. olivaceus.

Detection of LINE RT elements in the olive flounder (Paralichthys olivaceus) genome and expression analysis after infection with S. parauberis

Long interspersed nuclear elements (LINEs) are widely distributed in the vertebrate genome, and can be either beneficial or detrimental to the host genome. Here we identified three members of LINE RT elements in the olive flounder genome. They showed high amino acid sequence identity (89–99 %), and the sequences of LINE reverse transcriptase (RT) in olive flounder are closely related to those of coral grouper, European seabass, and three-spined stickleback. Real-time reverse transcription-polymerase chain reaction analysis indicated that expression of the OF (Olive flounder)-LINE Chr3-1 RT increased more in spleen than in other tissues after treatment with the pathogen Streptococcus parauberis. These data may form the basis for further studies on the function of retroelements in infected olive flounder.

Quantitative trait loci detection of Edwardsiella tarda resistance in Japanese flounder Paralichthys olivaceus using bulked segregant analysis

In recent years, Edwardsiella tarda has become one of the most deadly pathogens of Japanese flounder (Paralichthys olivaceus), causing serious annual losses in commercial production. In contrast to the rapid advances in the aquaculture of P. olivaceus, the study of E. tarda resistance-related markers has lagged behind, hindering the development of a disease-resistant strain. Thus, a marker-trait association analysis was initiated, combining bulked segregant analysis (BSA) and quantitative trait loci (QTL) mapping. Based on 180 microsatellite loci across all chromosomes, 106 individuals from the F1333 (♀: F0768 ×♂: F0915) (Nomenclature rule: F+year+family number) were used to detect simple sequence repeats (SSRs) and QTLs associated with E. tarda resistance. After a genomic scan, three markers (Scaffold 404-21589, Scaffold 404-21594 and Scaffold 270-13812) from the same linkage group (LG)-1 exhibited a significant difference between DNA, pooled/bulked from the resistant and susceptible groups (P <0.001). Therefore, 106 individuals were genotyped using all the SSR markers in LG1 by single marker analysis. Two different analytical models were then employed to detect SSR markers with different levels of significance in LG1, where 17 and 18 SSR markers were identified, respectively. Each model found three resistance-related QTLs by composite interval mapping (CIM). These six QTLs, designated qE1–6, explained 16.0%–89.5% of the phenotypic variance. Two of the QTLs, qE-2 and qE-4, were located at the 66.7 cM region, which was considered a major candidate region for E. tarda resistance. This study will provide valuable data for further investigations of E. tarda resistance genes and facilitate the selective breeding of disease-resistant Japanese flounder in the future.

Molecular cloning and sexually dimorphic expression of wnt4 in olive flounder (Paralichthys olivaceus).

WNT4 (wingless-type MMTV integration site family, member 4) is regarded as a key regulator of gonad differentiation in mammalians. However, the potential role of wnt4 in teleosts during gonad differentiation and development is still unclear. The full-length cDNA sequence of wnt4 in olive flounder (Paralichthys olivaceus) was obtained using RACE (rapid amplification of cDNA ends) technique. The wnt4 ORF contains 1059 nucleotides, encoding a protein with a signal peptide domain and a wnt family domain. Expression in tissues of adult flounders was analyzed by real-time RT-PCR. The results showed that wnt4 was widely expressed in multiple tissues of flounders, and the expression level was significantly higher in ovary than in testis. Then wnt4 expression pattern was investigated during gonadal differentiation period and at gonadal development stages (I-V). The results showed the expression levels were significantly higher in testis than in ovary during gonadal differentiation. Notably, wnt4 expression had a very significant increase before testis differentiation. At gonad different developmental stages, there was no expression signal at stage I or stage II, and the expression of wnt4 was much stronger in ovary than in testis at stage III and stage IV, followed by a faint expression in stage V in both sexes. Our results imply that cloned wnt4 could be wnt4a. It is a sex-related gene and its expression pattern in gonadal differentiation period of flounder is different from that in mammalians or other teleosts. Flounder wnt4 might play more important role in testis than in ovary during gonadal differentiation.