Program

Vibrio harveyi is a Gram-negative fish pathogenic bacterium. We performed transcriptome analysis of kidney and spleen in orange-spotted grouper (Epinephelus coioides) 1 and 2 days after Vibrio harveyi infection, using the Illumina sequencing platform. After de novo assembly, a total of 79,128 unigenes were detected with an N50 of 2511 bp. After alignments with sequences recorded in the major databases (NT, NR, Swiss-Prot COG, KEGG, Interpro and GO) based on sequence similarity, 61,208 (77.4%) of total unigenes could be annotated using at least one database. Comparison of gene expression levels between Vibrio harveyi and control group at each time points showed the differentially expressed genes (P < 0.05), a total of 7918 (5536 upregulated and 2282 downregulated genes) from head kidney at 1 dpi, 4260 (1444 upregulated and 2816 downregulated genes) from head kidney at 2 dpi, 7887 (4892 upregulated and 2995 downregulated genes) from spleen at 1 dpi and 8952 (7388 upregulated and 1564 downregulated genes) from spleen at 2 dpi. The differentially expressed genes were mainly annotated into signal transduction and immune system based on KEGG data base. The DEG were enriched in immune related pathway, NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, NF-kappa B signaling pathway and Jak-STAT signaling pathway. Additionally, we selected several DEGs and validated these expression level using qRT-PCR. These data in this study may provide a valuable source for further immune response research and strategy against Vibrio harveyi infection in teleosts.

In mammals, skeletal muscle strength greatly decreases with aging. This is known as sarcopenia, a decrease in muscle mass and a reduction in the regenerative ability associated with aging. Sarcopenia severely affects the quality of life and health status for elderly people because of decrease in motor ability and metabolism. In contrast, in fish skeletal muscle, the muscle fibers continue to increase throughout their lifespan and regenerative ability is maintained high. TGF-β familiy members, myostatin (MSTN) and GDF11, share the same receptor and negatively regulate muscle formation. Recently Egerman et al. (2015) reported that the mRNA levels of GDF11 markedly increased with aging in rat skeletl muscle, suggesting a direct association of MSTN/GDF11 signal with sarcopenia in mammals. Here, we examined the expression levels of MSTN and GDF11 with aging in zebrafish skeletal muscle.
We used zebrafish RW strain at 2 months- (immature), 7 months- (adolescent), 16 months- (mature), and 39 months-old ages (aged). Zebrafish has single GDF11 gene and two paralogous myostatin genes, MSTNa and MSTNb. We designed gene specific primers and examined MSTN and GDF11 expression levels by qPCR using the GAPDH gene as an internal control. As a result, MSTNb decreased significantly in aged zebrafish skeletal muscle. Similar tendency was also obaserbed with MSTNa and GDF11. Our results indicate that at least at the mRNA levels, the expression of GDF11 and MSTN did not increase with aging in zebrafish skeletal muscle in contrast to mammalian counterparts. The decrease in MSTN/GDF11 signal may involve the supression of sarcopenia in fish skeletal muscle.

In human, skeletal muscle strength declines greatly with aging. This is known as sarcopenia, a decrease in muscle mass and a reduction in regenerative ability associated with aging. On the other hand, in fish skeletal muscle, the number of muscle fibers is increased throughout their lifespan and regenerative ability is maintained. Such negligible senescence of fish skeletal muscle is important for better understanding fish growth and mammalian sarcopenia. However, its molecular mechanism is almost unknown. Here, we conducted transcriptome analysis on zebrafish skeletal muscle to elucidate the molecular basis of the negligible senescence of fish skeletal muscle.
Zebrafish at 2 months-old (immature), 7 months-old (adolescent), 16 months-old (mature), and 39 months-old (aged) were used. Skeletal muscle was collected from each growth stage and RNAseq was performed using the Hiseq2000 sequencer. In zebrafish muscle, the expression of the heat shock protein (HSP) genes increased with aging. Conversely, ribosomal protein genes decreased in aged muscle. It has been reported that the overexpression of HSPs or the inhibition of ribosomal proteins extended lifespan in various species. HSPs and ribosomal proteins are under the control of target of rapamycin (TOR) signal, a key regulator of aging in various organisms. Suppression of senescence by inhibition of TOR signal was considered to occur in aged zebrafish skeletal muscle. Actually, our RNA-seq data showed significant increase of ATF3 expression in aged zebrafish skeletal muscle. ATF3 is a transcription factor the expression of which is upregulated by supression of TOR signal. We also observed accerelated aging in a TOR signal-activated transgenic zebrafish.
Taken together, our results suggest that fluctuation in TOR signals is the molecular basis for the negligible senescence of fish skeletal muscle.

Growth hormone (GH) transgenic fish are of great commercial interest owing to their potential to shorten production cycles and increase food production. However, the effect of over-expression of the GH gene on fish health has not been intensively studied. To examine such effects, we produced GH transgenic zebrafish using GH cDNA of Mozambique tilapia. Overexpression of IGF1 (6 fold) revealed by qPCR indicated the induction of GH/IGF-I axis in GH transgenic larvae.
GH/IGF-I signaling pathways also include target of rapamycin (TOR), one of the Akt targets. TOR signaling is the master regulator of cell growth, autophagy, and aging. By Western blotting using phospho-S6 (Ser235/236) antibody, we found that TOR signaling pathway was elevated. Elevation of GH/IGF1/TOR pathway in GH transgenic larvae raises the question regarding the status of autophagy. Then, the reduction of autophagy was analyzed qualitatively by 2D image analysis using anti-LC3 antibody in larvae and quantitatively by Western blotting using the same antibody in adult fish muscle, because it is an indicator of the poor health status of GH transgenic fish. Western blotting using LC3 antibody is the most widely used assay for quantitatively analyzing autophagy, however, it has a technical challenge, which makes it difficult to assess small changes in LC3 at the protein levels accurately. Here we show that 3D image analysis using Fiji software after LC3-whole mount immunostaining in zebrafish embryos will be used as a rapid, quantitative assay for precise LC3 puncta counting. And for evaluation of autophagic flux, it was used with presence and absence of lysosomal degradation. Using 3D image analysis, we found that autophagy is reduced in GH transgenic fish after addition of 3-methyladenine in a rapid, cheap and accurate way.

The low production of hypotaurine but a significantly high taurine production in common carp led to the hypothesis that this species utilizes an alternative pathway other than the cysteine sulfinic acid pathway which is commonly responsible for taurine production in animals.
The ADO cloned in common carp consists of 790 nucleotide bases with 260 deduced amino acid sequence having two identified open reading frames. The conserve domain was the DUF1637 which has a conserved tyrosine and cysteine residues and the presence of three predicted N-glycosylation sites.
Phylogenetic analysis using Joint neighboring method indicated that ADO in common carp is more advanced in evolution compared to Sinocyclocheilus rhinocerous, Danio rerio and Ictalurus punctatus.
ADO was present in hepatopancreas, brain, gills, intestines and muscles of common carp. The hepatopacreas had a significantly high gene expression level among the organs examined. The results suggest that ADO is present in common carp and is possibly an important enzyme being utilized by common carp for taurine synthesis.

In this study, we used CRISPR/Cas9 targeted genome editing technology to knock out (KO) two distinct myostatin genes, mstna and mstnb in zebrafish to understand their roles in muscle growth and unknown functions due to their extended expression in non-muscle tissues. We had obtained F2 offspring of mstna KO、mstnb KO and mstna/mstnb double KO zebrafish lines to compare their phenotypes with wild-type zebrafish. We found that the muscle growth was most significantly enhanced in mstna & mstnb (mstna/b) double KO zebrafish at 90 dpf, which exhibiting obvious phenotype with bulging muscles on the back. The muscle growth of mstnb KO zebrafish is better than that of mstna KO zebrafish and wild-type zebrafish. The muscle histological analysis show moderate hyperplasia muscle cell in subcutaneous in mstna/b double KO zebrafish. Mstnb KO zebrafish section shows muscle fibers hyperplasia evenly with smaller areas compared with the wild-type control fish. However, the 150 dpf mstna/b double KO and mstna KO zebrafish showed just slightly higher body weight than WT, but mstnb KO increased to 1.4-fold in body weight. Compare morphology of each groups, some mstna/b double KO zebrafish got rickets and have high mortality rate in adult fish and fertilize egg. The muscle section shows marvelous hyperplasia in mstna/b double KO zebrafish. Conversely, the originally smaller hyperplasia mstnb muscle fiber grow bigger, turns to be hypertrophy. According to our results, we speculate mstna/b double knockout affect not only myogenesis but also aging, reproduction and immunity. Zebrafish mstnb is the major negative regulator involved in muscle growth than mstna. The functions of mstna and mstnb involved in aging, reproduction and even immunity can be compensated each other in mstna or mstnb gene single KO zebrafish. Our study suggest that new double muscle teleost fish strains can be achieved by major mstnb gene knockout with CRISPR/Cas9 technology to obtain obvious muscle enhancement without observed side effect.

Recent progresses in genome editing using programmable nucleases have provided advanced gene targeting in various organisms. This is also received considerable attention as a new breeding technology in the field of aquaculture. Here, we report genome editing using transcription activator-like effector nuclease (TALEN) in kawakawa, which is a new tuna species on the market as an aquaculture species in Japan. In this study, we have selected gene, tyrosinase (tyr), which is responsible for albinism as a target for the genome editing.
Two TALENs, tyr-B and tyr-D, were designed and constructed using Platinum TALEN. Morpholino knockdown of tyr (tyrMO) was used to identify the gene underlying the phenotype changes. Embryos were injected the TALEN RNAs or tyrMO one- to two- cell stage and were incubated at 24˚C. Phenotype changes of pigmentation were observed periodically with development. Larvae at 90 hpf from control and experimental groups were collected and subjected to heteroduplex mobility assays (HMA) to confirm changes in target genome sequences.
Two types of pigment cells were found in embryos at 24 hpf and after. One type was confirmed as melanocytes by injecting tyrMO. Another type was unidentified pigment cells with brown color. Melanin pigmentation was evident in the melanophore on the body and retinal pigment epithelium (RPE) which were apparent at 70 hpf in control fish. Morphants showed that complete melanin depletion until 48 hpf and gradually recovered melanin pigmentation in the RPE after 70 hpf. TALEN mutants showed different degree of phenotype changes after 70 hpf. Mosaic pattern of RPE and no melanin pigments on the body were found in the TALEN-injected fish. HMA and sequence analysis showed that indel mutations were detected at the target genome.
In conclusion, we confirmed that TALEN was effective for genome editing in kawakawa.

Organic chemicals derived from artificial sources, including industrial effluents, domestic sewage and farm wastes, attract wide attention as harmful contaminants in the global environment. They are major pollutants in water and have adverse effect on aquatic organisms and humans. Biondicators which indicate the pollutant level in the water should be established to ensure the safety of the consumption of water and aquatic organisms. Here, we report the attempt to establish a bioindicator strain of Javanese medaka, Oryzias javanicus, known as an excellent vertebrate model that can be reared in seawater. Using the genome editing technique, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9 system, we knocked out the cytochrome P450 family 1A (CYP1A) gene, one of the most important genes for metabolisms of various organic chemicals. Before starting knockout experiments, CYP1A cDNA sequences of three O. javanicus strains originated from different locations were determined to detect the existence of single nucleotide polymorphisms (SNPs) sites, which may affect the efficiency of the knockout process. Reverse transcription PCR (RT-PCR) was also carried out to analyse tissue-specificity of the gene expression. Besides, tolerance and response against a representative organic pollutant, phenanthrene was also tested. Subsequently, single guide RNA (sgRNA) was designed using the site where no SNP detected, and the sgRNA and Cas 9 RNA were microinjected into fertilized eggs. From the injected eggs, many adult fish with mutated CYP1A gene were obtained. We will report establishment process of homozygous strains for mutated CYP1A gene, and their response to phenanthrene and other chemicals.

The basic knowledge on molecular mechanisms and functional involvement of reproduction-related gene/proteins is necessary for better understanding of the reproductive maturation of the giant tiger shrimp Penaeus monodon. Proteomics of nuclear and nuclear membrane proteins in ovaries of domesticated and wild broodstock of P. monodon were studied using GeLC-MS/MS. In total, 724 known proteins were identified. Localization of these proteins were searched and 89 (e.g. cytochrome b5, cyclic AMP-regulated protein like protein, semaphorin-1A) and 99 (e.g. importin-β, ran gtpase-activating protein, cell division cycle 2 and cyclin dependent kinase 2) proteins were classified as those integrated to membrane and nuclear proteins, respectively. The full-length cDNAs of cytochrome b5 (PmCytB5; 1539 bp in length containing an ORF of 432 bp deducing to 143 amino acids), cyclic AMP-regulated protein like protein (PmcAMP-RLP; 1272 bp, ORF of 435 bp, 144 amino acids) and cyclin dependent kinase 2 (PmCdk2; 1763 bp, ORF of 921 bp, 306 amino acids) were isolated. Expression levels of these genes during ovarian development of wild intact- and eyestalk-ablated P. monodon adults were examined. Eyestalk ablation resulted in significant lower expression of PmCytB5 and PmCdk2 (P < 0.05) but greater expression level of PmcAMP-RLP (P < 0.05) suggesting that unilateral eyestalk ablation affected the expression of these reproduction-related transcripts. In addition, effects of serotonin (5-HT) administration in domesticated 14-month-old P. monodon were evaluated. Results indicated that the expression level of PmcAMP-RPL was stimulated by 5-HT administration. In contrast, The PmCytB5 level was not significant altered after serotonin administration but its expression level was significantly induced at 72 hour post progesterone injection. Results in this study indicated the potential of proteomic studies and gene expression analysis for identification of proteins/genes functionally involved in ovarian development of P. monodon.

Two full-length cDNAs of farnesoic acid O-methyltransferase (FAMeT) in Penaeus monodon were identified. They were 1296 and 1311 bp containing ORFs of 828 (PmFAMeT-s) and 843 (PmFAMeT-l) bp deducing to 275 and 280 amino acids. Single nucleotide polymorphisms (SNPs) in PmFAMeT of 14-month-old P. monodon (N = 65) of the 5th generation of domesticated P. monodon were examined by DNA sequencing. Six SNPs were found. Two SNPs (positions c.487+g.10 and c.487+g.174+c.38) were significantly associated with the average ovarian weight of examined shrimp (P < 0.05). In addition, the relative expression level of ovarian PmFAMeT mRNA in shrimp with T/Cc.487+g.10 and T/Tc.487+g.10 was also significantly different (P < 0.001). In addition, association between SNP genotypes and growth parameters of 5-month-old domesticated juveniles were examined. Analysis of its nucleotide sequence revealed that SNP at positions c.487+g.10 and c.487+g.174+c.8 can be restricted with HpyCH4V. Interestingly, two positions of the A/G SNP are found as a result, the predicted genotypes can be deduced for either each locus or from both positions as composite SNP genotypes (diplotypes). PCR-RFLP was carried out against genomic DNA of in 23 full-sib families of the 7th generation of domesticated P. monodon cultured in the concrete ponds (N = 323). At position c.487+g.10, significant differences between growth parameters of juveniles having different genotpyes were observed where juveniles with a T/Cc.487+g.10 genotype showed a greater average body weight and total length than those with the T/C c.487+g.10 and C/C c.487+g.10 genotype (P < 0.05). At position c.487+g.174+c.8, significant differences between the body weight of juveniles having different genotpyes were also observed where juveniles with A/Ac.487+g.174+c.8 and A/Gc.487+g.174+c.8 had a greater average body weight and total length than those with G/Gc.487+g.174+c.8 (P < 0.001). Similarly, significant differences in growth-related parameters were observed in juveniles carrying different diplotypes (P < 0.001).

Molecular markers that allow selection of juveniles and broodstock with a high breeding value for growth rates are useful for the shrimp industry. Here, the full-length cDNAs of cyclin C (PmCycC) in Penaeus monodon were isolated. Polymorphism of PmCycC, cell division cycle 25 (PmCdc25) and molt-inhibiting hormone (PmMIH) in 3-month-old juveniles of domesticated P. monodon (SNP3A sample) was identified by DNA sequencing. Juveniles having different SNP genotypes of PmCycC (C/T134 > C/C134), PmCdc25 (A/C228 > C/C228) and PmMIH (G/T217 > G/G217) exhibited significantly different average body weight and total length (P < 0.05). Association between candidate SNP markers in these genes and growth-related parameters of domesticated P. monodon were further examined in 12 full-sib families of the 7th generation (5-month-old) cultured in three concrete ponds (G7-1, G7-2 and G7-3 samples). Initially, SNP of these gene segments was determined by DNA sequencing. Subsequently, indirect detection methods (gel-based Bi-PASA, real-time PCR-based PASA and/or PCR-RFLP) were developed for detection of SNP in PmCdc25 (A>C228), PmCycC (C>T134) and PmMIH (G>T217). Results were also consistent for the association between SNP of these genes and growth-related parameters (average body weight and total length) of examined shrimp (P < 0.05). In addition, expression levels of PmCdc25 and PmCycC in hepatopancreas and pleopods of the G7-1 sample were analyzed to determine whether juvenile with different SNP genotypes possessed different expression level or not. Results showed a trend of different expression of these genes in juveniles with different SNP genotypes. Taken the information together, results in this study suggested the potential application of growth-related SNPs for breeding programs of domesticated P. monodon.

With the emergence of several diseases in shrimp aquaculture, immunostimulants have received increasing attention for its positive impacts on immunity and disease resistance. Here, we investigated the effect of 5-aminolevulinic acid (5-ALA), a non-protein amino acid reported to enhance immunity in a wide range of organisms including humans, livestock animals and plants. Using microarray analysis, we compared the expression profile between Litopenaeus vannamei fed with experimental diet (containing 10ppm 5-ALA) and the control (fed with basal diet) after two weeks of feeding. We then validated the results for selected differentially expressed genes (DEGs) using qPCR. After the feeding trial, we conducted challenge tests with three pathogens (white spot syndrome virus, Vibrio parahaemolyticus and Fusarium solani) and recorded mortality rates daily up to two weeks.
Out of 15,745 L. vannamei putative genes on the microarray, 101 genes were differentially expressed in the hepatopancreas more than 4-folds between 5-ALA-fed and control groups. By comparison with functionally annotated homologous genes, many of these DEGs were shown to have molecular functions such as ion binding, signal transduction and oxidoreductase activities. qPCR results have confirmed upregulation of immune-related genes such as lysozyme, c-type lectin, peritrophin-A and nitric oxide synthase in 5-ALA-fed group. For the challenge tests, 5-ALA-fed group had statistically higher survival rate compared to the control for V. parahaemolyticus challenge while no significant differences were observed for the other tests.
The findings of this study suggested that 5-ALA increased the expression of immune-related genes relative to the control. Challenge tests also indicated that 5-ALA may have an important role in bacterial infection, but not for viral and fungal infections. Although current results showed promising effects of 5-ALA on shrimp immunity, further studies are necessary to determine effects of longer feeding period and different 5-ALA concentrations.

Penaeidins are a family of antimicrobial peptides constitutively produced and stored in the hemocytes of penaeid shrimps. MjPen-II, a penaeidin isolated from kuruma shrimp (Marsupenaeus japonicus) belonging to Penaeidin subfamily II, plays an important role in the host defense system of shrimp. A previous study has reported antibacterial activity of MjPen-II but no report has validated whether it is also responsive to other pathogens like white spot syndrome virus (WSSV). Furthermore, it is also unclear which type of hemocytes produce and store MjPen-II. Thus, we identified MjPen-II in hemocytes after WSSV challenge and investigated MjPen-II localization using antiserum against MjPen-II.
mRNA expression of MjPen-II in six tissues (hemocytes, gills, hepatopancreas, stomach, intestine and lymphoid organ) were measured at 0h, 6h, 24h and 72h post-challenge with WSSV. Recombinant protein of MjPen-II (rMjPen-II) was produced in Escherichia coli, then rabbit antiserum against purified rMjPen-II was prepared. Antiserum specificity against MjPen-II in hemocytes was confirmed by Western blotting. In addition, localization of MjPen-II in hemocytes was detected by immunostaining.
MjPen-II mRNA transcripts were significantly downregulated in five tissues except intestine after 24h post-challenge. Moreover, a single specific band of 15 kDa, which corresponds to the predicted molecular mass of MjPen-II, was detected in hemocytes by Western blotting. From immunostaining result, MjPen-II was observed in the cytoplasm and positive signals were observed only in granule-containing hemocytes. In contrast, no signal was observed in hyaline cells, which were smaller and had no noticeable intracytoplasmic granules. These results suggested that this antibody could potentially separate granular from non-granular cells. Further studies are necessary to investigate downregulation and localization of MjPen-II after WSSV challenge.

White spot syndrome virus (WSSV) is a viral pathogen causing massive economic losses to the shrimp aquaculture industries worldwide. WSSV is a double-stranded DNA virus that infect a wide range of crustaceans, classified as the sole member of the genus Whispovirus in the monotypic family Nimaviridae. The lack of genetic information on related viruses obscures the evolutionary history of WSSV.
In previous work, we have shown that the genome of kuruma shrimp Marsupenaeus japonicus, a typical WSSV host, harbors large repetitive DNA segments containing clusters of sequences showing similarity to WSSV genes at the deduced amino acid level. We suspected the WSSV-like sequences represent the evidence of an ancient viral integration event that took place in the progenitor of M. japonicus. Further, characterization of other nimavirus genomes endogenized in crustacean genomes would enable us to trace the evolutionary history of nimaviruses in a geological time scale.
Here, we report on at least four instances of endogenized nimavirus genomes integrated as repetitive elements in the genome of six penaeid shrimps and two sesarmid crabs. The nimavirus genome initially found in M. japonicus seems to have been endogenized into the genome of the common ancestor of M. japonicus and Litopenaeus vannamei, suggesting that the emergence of Nimaviridae dates to at least 88 million years ago. Penaeus monodon harbors two distinct nimavirus genomes. Metapenaeus ensis genome contains a nimavirus genome closely related to extant WSSV. The genome of two Asian sesarmid crabs harbor a nimaviral genome putatively orthologous to the one originally found in a closely related species in Central America.
These findings indicate that there is a long, intimate relationship between Nimaviridae and decapod crustaceans. Closer analyses of the “fossilized” nimavirus genomes will help to understand the evolutionary dynamics of nimaviruses and the molecular biology of WSSV infection.

RNA-seq was used to compare the transcriptomic response of hepatopancreas in juvenile Litopenaeus vannamei fed dietary ulvan or fucoidan at 2 g kg¹ for 2 weeks. A total of 119.38 million reads were obtained from the hepatopancreas of L. vannamei. After quality trimming and adapter clipping, a total of 115.86 million high quality reads remained and were assembled to 107,730 unigenes. Among the annotated and predictable sequences, a total of 38,167 (35.42%), sequences were unambiguous alignments relative to the reference after BLASTx against NR. For function classification and pathway assignment, 36,564 (33.94%) unigenes were categorized to three Gene Ontology (GO) categories, 18,492 (17.16%) were classified to 26 Clusters of Orthologous Groups (KOG); unigenes were assigned to 5 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Between Ulvan and Control group 53 differentially expressed genes (DEGs) were identified; between Fucoidan and Control group 77 DEGs. Unigenes were characterized to be involved in Hippo signaling pathway, MAPK signaling pathway, GABAergis synapse and endometrial cancer. Conclusion/Significance: This is the first transcriptome analysis of components relating to pathways in L. vannamei as a result of feeding a potential immunostimulant in the form of sulfated polysaccharides ulvan and fucoidan.

Olive flounder (Paralichthys olivaceus) is an important commercially cultured marine flatfish in China. The female flounder shows significant advantages in growth and individual size than the male one, and its sex ratio is sensitive to temperature or exogenous hormone exposures in the period of gonadal differentiation. This study comprehensively clarified the mechanism of flounder gonadal differentiation from the perspective of transcriptome, gene expression, epigenetics, endocrinology, and gene regulation. RNA-seq technology was firstly used to investigate gonadal transcriptomes of flounder. The differentially expressed genes between ovary and testis were identified, which could provide candidate genes for further studies of molecular mechanism of flounder sex differentiation. Among sex-related genes, cyp19a, encoding cytochrome P450 aromatase，exhibits significant sex-dimorphic expression pattern and plays an important role in fish gonadal differentiation and development. We comprehensively analyzed the expression levels of cyp19a and its transcription factors, such as foxl2, nr5a2, nr0b1 and dmrt1, and their promoter methylation level variation during gonadal differentiation period under high temperature or exogenous sex steroid hormones treatment. The effect of these genes on gonadal differentiation direction was explored combining the endogenous sex hormone level variation of flounder. The spatial expression patterns of foxl2, nr5a2, nr0b1 in gonad and their regulation on cyp19a expression were also demonstrated. And the effect of promoter fully methylation on cyp19a expression was investigated to explore its regulation mechanism. This research would provide basic data for further investigation of environmental factors on sex phenotype formation in flounder and other fishes.