Tissues Of Flounder Research Articles

Characterization of CD40+ leukocytes in flounder (Paralichthys olivaceus) and its response after Hirame novirhabdovirus infection and immunization

CD40 is a crucial signal mediating factor in T-dependent B cell responses and involved in many aspects of cellular and humoral immunity. In this paper, recombinant protein of CD40 in flounder (Paralichthys olivaceus) and its antibodies (Abs) were produced, native CD40 molecules in flounder tissues were identified, then the CD40+ leukocytes in T/B lymphocytes were characterized, and the variations of CD40+ leukocytes in flounder after Hirame novirhabdovirus (HIRRV) infection and immunization were investigated, respectively. The results showed that the Abs could specifically recognize native flounder CD40 molecule at 32 kDa. The proportions of CD40+ leukocytes were varied by flounder tissues. CD40+/IgM+ B lymphocytes, CD40+/CD4-1+ T lymphocytes, CD40+/CD4-2+ T lymphocytes and CD40+/CD8+ T lymphocytes in peripheral blood leukocytes (PBLs) were 1.18 ± 0.27%, 0.69 ± 0.17%, 0.75 ± 0.14% and 0.25 ± 0.14%; were 2.80 ± 0.32%, 0.71 ± 0.19%, 0.88 ± 0.23% and 0.33 ± 0.17% in spleen; 4.11 ± 0.47%, 0.92 ± 0.18%, 1.09 ± 0.17% and 0.9 ± 0.17% in head kidney; 1.92 ± 0.39%, 1.02 ± 0.23%, 1.33 ± 0.38% and 0.67 ± 0.24% in intestine; 1.24 ± 0.36%, 1.21 ± 0.24%, 1.70 ± 0.3% and 0.97 ± 0.21% in gill, respectively. The percentages of CD40+ leukocytes in PBLs were significantly increased in both HIRRV infection and immunization groups, and reached their peak levels at 3rd day with 5.70 ± 0.16% and 6.40 ± 0.13%, respectively. Concluded with our previous study, these data first reported that CD40 molecules were expressed on both B and T lymphocytes in teleost, and had a coordination with T and B lymphocytes in immune responses.

Characterization of UDP-Activated Purinergic Receptor P2Y6 Involved in Japanese Flounder Paralichthys olivaceus Innate Immunity

Uridine 5’-diphosphate (UDP)-activated purinergic receptor P2Y6 is a member of a G-protein-coupled purinergic receptor family that plays an important role in mammalian innate immunity. However, the role of the P2Y6 receptor (P2Y6R) in fish immunity has not been investigated. In this report, we characterized a P2Y6R gene from Japanese flounder (Paralichthys olivaceus) and examined its role in fish innate immunity. Sequence analysis reveals that the Japanese flounder P2Y6R protein is conserved and possesses four potential glycosylation sites. Quantitative real-time RT-PCR analysis shows that P2Y6R is broadly distributed in all examined Japanese flounder tissues with dominant expression in the liver. In addition, P2Y6R gene expression was up-regulated in head kidney macrophages (HKMs) upon lipopolysaccharides (LPS) and poly(I:C) stimulations but down-regulated by LPS challenge in peripheral blood leukocytes (PBLs). Furthermore, pharmacological inhibition of the endogenous P2Y6 receptor activity by the potently selective P2Y6R antagonist, MRS 2578, greatly up-regulated pro-inflammatory cytokine interleukin (IL)-1β, IL-6 and TNF-α gene expression in PBL cells treated with UDP. Moreover, LPS- and poly(I:C)-induced gene expression of IL-1β and TNF-α in Japanese flounder PBL cells was attenuated significantly by inhibition of P2Y6R activity with antagonist MRS 2578. Collectively, we, for the first time, showed the involvement of functional purinergic P2Y6R in fish innate immunity.

Methylation Status of the Follistatin Gene at Different Development Stages of Japanese Flounder (Paralichthys olivaceus)

Follistatin (Fst) is a hyperplasia factor that plays a crucial role in muscle development. DNA methylation, a significant process, regulates gene expression. The aim of our study is to examine the DNA methylation and expression patterns of Fst gene at five different development stages of Japanese flounder (stage A, 7 dph; stage B, 90 dph; stage C, about 180 dph; stage D, about 24 months; stage E, about 36 months). The muscle tissue of Japanese flounder was obtained at different development stages in this experiment. DNA methylation levels in the promoter and exon 2 of Fst were determined by bisulfite sequencing, and the relative expression of the Fst gene at the five stages was measured by quantitative PCR. The results showed that the lowest methylation level was at stage A and the highest methylation level was at stage B. Moreover, the highest expression level of the Fst gene was observed at stage A. The mRNA abundance was negatively correlated with DNA methylation level. Three CpG islands in the promoter region and three CpG islands in exon 2 of Fst were found in the binding sequence of the putative transcription factor. These results offered a theoretical basis for the mechanism of Fst gene regulation to muscle development at different development stages.

Functional characterization of purinergic P2Y2 and P2Y12 receptors involved in Japanese flounder (Paralichthys olivaceus) innate immune responses

G-protein-coupled P2Y receptors activated by extracellular nucleotides play important roles under different physiological and pathophysiological conditions in mammals. To investigate the immunological relevance of P2Y receptors in fish, we identified and characterized the P2Y2 and P2Y12 receptors in Japanese flounder Paralichthys olivaceus. The P. olivaceus P2Y2 and P2Y12 receptors harbor seven transmembrane domains but share only 24% sequence identity. Real-time PCR analysis revealed the constitutive but unequal mRNA expression pattern of P2Y2R and P2Y12R in normal Japanese flounder tissues with the dominant expression of P2Y2R in head kidney and blood and P2Y12R in hepatopancreas. In addition, the expression of P2Y2 and P2Y12 receptors was markedly modulated by PAMPs stimulation and Edwardsiella tarda infection. Furthermore, blockage of P2Y12R potently increased ADP-activated pro-inflammatory cytokine IL-1beta gene expression in the head kidney macrophages (HKMs). Moreover, inhibition of P2Y2 and P2Y12 receptor activity with their respective potent antagonists significantly altered some of the LPS-induced pro-inflammatory cytokine gene expression in the HKMs. However, blockade of P2Y12R did not affect the poly(I:C)-induced pro-inflammatory cytokine gene expression examined in the HKMs. Collectively, we have for the first time reported the role of purinergic P2Y2 and P2Y12 receptors in fish innate immunity. Our findings have also addressed the importance of extracellular ATP and its metabolites in fish innate immune responses.

Genomic Analysis of miR-21-3p and Expression Pattern with Target Gene in Olive Flounder.

MicroRNAs (miRNAs) act as regulators of gene expression by binding to the 3’ untranslated region (UTR) of target genes. They perform important biological functions in the various species. Among many miRNAs, miR-21-3p is known to serve vital functions in development and apoptosis in olive flounder. Using genomic and bioinformatic tools, evolutionary conservation of miR-21-3p was examined in various species, and expression pattern was analyzed in olive flounder. Conserved sequences (5’-CAGUCG-3’) in numerous species were detected through the stem-loop structure of miR-21-3p. Thus, we analyzed target genes of miR-21-3p. Among them, 3’ UTR region of PPIL2 gene indicated the highest binding affinity with miR-21-3p based on the minimum free energy value. The PPIL2 gene showed high expression levels in testis tissue of the olive flounder, whereas miR-21-3p showed rather ubiquitous expression patterns except in testis tissue, indicating that miR-21-3p seems to control the PPIL2 gene expression in a complementary repression manner in various tissues of olive flounder. Taken together, this current study contributes to infer the target gene candidates for the miR-21-3p using bioinformatics tools. Furthermore, our data offers important information on the relationship between miR-21-3p and target gene for further functional study.

Mercury, Fatty Acids Content and Lipid Quality Indexes in Muscles of Freshwater and Marine Fish on the Polish Market. Risk Assessment of Fish Consumption.

Mercury content and fatty acids in muscles of Perca fluviatilis L. (European perch), Leuciscus idus L. (ide), Cyprinus carpio L. (European or common carp), Oncorhynchus mykiss Walb. (rainbow trout), Platichthys flesus L. (European flounder). and Clupea harengus L. (bream) from the Polish market were investigated. The total mercury was processed with AAS. The fatty acids were analyzed by gas chromatography. The concentration of mercury in muscles varied from 0.006 to 0.138 mg/kg and decreased as follows: perch ≈ ide > flounder > herring ≈ bream ≈ rainbow trout > carp (p ≤ 0.05). There were only significant positive correlations between body weight and mercury content in muscle tissue of carp (r = 0.878), flounder (r = 0.925) and herring (r = 0.982) (p ≤ 0.05). The atherogenic index (AI), thrombogenicity index (TI) and flesh-lipid quality index (FLQ) were calculated as follows 0.33–0.70 (IA), 0.16–0.31 (IT) and 13.01–33.22 (FLQ). Hypocholesterolemic (OFA) and hypercholesterolemic fatty acids (DFA) in muscles of fish ranged from 18.26 to 23.01 and from 73.91 to 78.46, respectively. In most cases, there were not significant correlations between size (body weight and total length) and fatty acids in the muscles of the examined fish (p > 0.05). The Target Hazard Quotient (THQ) values were below 1, which shows that there is no non-carcinogenic health risk to the consumer by consuming the examined fish.

Comparative analysis of dual specificity protein phosphatase genes 1, 2 and 5 in response to immune challenges in Japanese flounder Paralichthys olivaceus

Dual-specificity MAP kinase (MAPK) phosphatases (DUSPs) are well-established negative modulators in regulating MAPK signaling in mammalian cells and tissues. Our previous studies have shown the involvement of DUSP6 in regulating innate immunity in Japanese flounder Paralichthys olivaceus. In order to gain a better understanding of the role of DUSPs in fish innate immunity, in the present study we identified and characterized three additional DUSP genes including DUSP1, 2 and 5 in P. olivaceus. The three Japanese flounder DUSP proteins share common domain structures composed of a conserved N-terminal Rhodanase/CDC25 domain and a C-terminal catalytic phosphatase domain, while they show only less than 26% sequence identities, indicating that they may have different substrate selectivity. In addition, mRNA transcripts of all the three DUSP genes are detected in all examined Japanese flounder tissues; however, DUSP1 is dominantly expressed in spleen while DUSP2 and 5 are primarily expressed in skin. Furthermore, all the three DUSP genes are constitutively expressed in the Japanese flounder head kidney macrophages (HKMs) and peripheral blood leucocytes (PBLs) with unequal distribution patterns. Moreover, all the three DUSPs gene expression was induced differently in response to the LPS and double-stranded RNA mimic poly(I:C) stimulations both in the Japanese flounder HKMs and PBLs, suggesting an association of DUSPs with TLR signaling in fish. Taken together, the co-expression of various DUSPs members together with their different responses to the immune challenges indicate that the DUSP members may operate coordinately in regulating the MAPK-dependent immune responses in the Japanese flounder.

Two skin cell lines from wild-type and albino Japanese flounder (Paralichthys olivaceus): establishment, characterization, virus susceptibility, efficient transfection, and application to albinism study.

In order to provide an applicable cell platform to study fish pathology and skin pigmentation, two cell lines derived from skin tissues of wild-type and albino Japanese flounder were established and named JFSK_wt and JFSK_alb, respectively. These two cell lines were cultured for 45 passages within approximately 300days. JFSK_wt and JFSK_alb cells were maintained in Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mixture (DMEM/F12) supplemented with antibiotics, fetal bovine serum (FBS), 2-mercaptoethanol (2-Me), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and basic fibroblast growth factor (bFGF). The optimal growth temperature for JFSK_wt and JFSK_alb cells was 24°C, and microscopically, the two cell lines were composed of fibroblast-like cells. Chromosomal analysis revealed that JFSK_wt and JFSK_alb cells had an identical diploid karyotype with 2n = 48t. Results of viral inoculation assays revealed that both cell lines shared similar patterns of viral susceptibility to nervous necrosis virus (NNV). High transfection efficiency was observed in JFSK_wt and JFSK_alb cells transfected with a pEGFP-N3 reporter plasmid and Cy3-siRNA. The detection of dermal marker Dermo-1 showed that these two cells were both derived from the dermis. Finally, three genes involved in the melanogenesis pathway, including adenylate cyclase type 5 (adcy5), microphthalmia-associated transcription factor (mitf), and endothelin B receptor (ednrb), were downregulated in JFSK_alb versus JFSK_wt cells. Thus, the two cell lines, sampled from skin tissue of wild-type and albino Japanese flounder will be not only helpful for fish pathogen research but also beneficial for albinism-related gene function studies.

Interferon-induced protein 56 (IFI56) is induced by VHSV infection but not by bacterial infection in olive flounder (Paralichthys olivaceus)

Interferon-inducible protein 56 (IFI56, also known as ISG56/IFIT1, interferon-induced protein with tetratricopeptide repeats 1) is strongly induced in response to interferon and a potent inhibitor of viral replication and translational initiation. Here, we describe the identification of IFI56 (OfIFI56) in olive flounder, its characteristic features, and expression levels in various tissues before and after viral hemorrhagic septicemia virus (VHSV) infection. The full-length OfIFI56 sequence was identified from rapid amplification of cDNA ends PCR. The complete coding sequence of OfIFI56 is 1971 bp in length and encodes 431 amino acids. The putative OfIFI56 protein has multiple tetratricopeptide (TPR) motifs, which regulate diverse biological processes, such as organelle targeting, protein import, and vesicle fusion. Based on sequence analysis, the Larimichthys crocea IFI56 protein (61%) had the highest sequence homology to OfIFI56. In healthy olive flounder, OfIFI56 mRNA expression was detected in many tissues such as intestine, gill, head kidney, heart, spleen, and trunk kidney tissues. After VHSV challenge, OfIFI56 mRNA was significantly up-regulated in these tissues. Additionally, OfIFI56 expression was induced by poly I:C but not by Streptococcus parauberis and S. iniae infection or lipopolysaccharide injection in kidney and spleen tissues of olive flounder. These results demonstrate that piscine OfIFI56 expression is not induced by bacterial infection but is selectively induced by viral infection, especially VHSV, and that OfIFI56 may play an important role in the host response against VHSV infection.

Metabolite and gene expression responses in juvenile flounder Paralichthys olivaceus exposed to reduced salinities

Seawater salinity is one of the most important changeable environmental factors influencing the behavior, survival, growth and production of marine organisms. In this work, metabolite and gene expression profiles were used to elucidate the biological effects of reduced salinities in juvenile flounder Paralichthys olivaceus. Metabolic profiling indicated that both reduced salinities (23.3‰ and 15.6‰) enhanced proteolysis and disturbed osmotic regulation and energy metabolism in juvenile flounder P. olivaceus. Furthermore, the low salinity (15.6‰) enhanced anaerobic metabolism indicated by the elevated lactate in flounder tissue extracts. Gene expression profiles exhibited that reduced salinities could induce immune stress and oxidative stress and disturb energy metabolism in juvenile flounder P. olivaceus. In addition, reduced salinities might promote the growth and gonadal differentiation in juvenile flounder P. olivaceus.

Expression analysis of three immune genes Interferon-gamma, Mx and Interferon regulatory factor-1 of Japanese flounder ( Paralichthys olivaceus )

ABSTRACT IFN-γ (Interferon-gamma), Mx and IRF-1 (Interferon regulatory factor-1) are main immune-related genes and they play important roles in the innate immune system of vertebrates. In this study, the expression level of the three immune-related genes in twelve tissues of normal adult Japanese flounder was detected using semi-quantitative RT-PCR. Thirteen time points (3h, 6h, 9h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, 108h, 120h) were selected to analyze the expression of IFN-γ, Mx and IRF-1 in spleen, head kidney, liver of Japanese flounder infected with Lipopolysaccharide (LPS). The Japanese flounder IFN-γ, Mx and IRF-1 genes were differently expressed in these tissues and had high expression levels in classical fish immune organs like spleen and head kidney. It was found that the highest expression levels of the Japanese flounder IFN-γ were detected at 24h in spleen, 36h in head kidney and 48h in liver after challenge with LPS. Interestingly, the highest expression levels of Mx in spleen and head kidney were both at 36h and IRF-1 in spleen and liver were both at 24h. The highest expression level of Mx in liver was at 48h and IRF-1 in head kidney was at 12h. The study provides a basis for further research on immune mechanism of IFN-γ, Mx, IRF-1 and the production of recombinant IFN-γ, Mx or IRF-1 used in Japanese flounder cultivation in future.

Expression of phospholipase C β1 in olive flounder (Paralichthys olivaceus) following external stress stimulation

In this study, to clarify the function of PoPLC-β1, in response to stress challenge, we examined the PoPLC-β1 expression pattern in response to external stress (pathogen-associated molecular pathogen challenge and environmental challenge including temperature and salinity). PoPLC-β1 expression analysis of tissue from olive flounder showed that the messenger RNA (mRNA) was predominantly expressed in the brain, heart, eye, liver, spleen, and stomach. We also tested the mRNA expression of the PoPLC-β1 in the spleen and kidney of olive flounder by RT-PCR and real-time PCR following stimulation with lipopolysaccharide (LPS), concanavalin A (ConA), or polyinosinic:polycytidylic acid (PolyI:C) and compared with the inflammatory cytokines IL-1b and IL-6 in the stimulated flounder tissues. Each of the spleen and kidney and mRNA transcripts of PoPLC-β1 were increased 30- and 10-fold than normal tissue at 1–6 h post injection (HPI) with PolyI:C when the expression of PoPLC-β1 transcript was similar to LPS and ConA. We also tested the expression of PoPLC-β1 in response to temperature and salinity stress. The expression of PoPLC-β1 also was affected by temperature and salinity stress. Our results provide clear evidence that the olive flounder PLC-β1 signal pathways may play a critical role in immune function at the cellular level and in inflammation reactions. In addition, PLC-β1 appears to act as an oxidative-stress suppressor to prevent cell damage in fish.

Antigen uptake and expression of antigen presentation-related immune genes in flounder (Paralichthys olivaceus) after vaccination with an inactivated Edwardsiella tarda immersion vaccine, following hyperosmotic treatment.

Antigen uptake is a critical process for activation of the immune system, and therefore the ability to enhance antigen uptake is a primary consideration in the development of an immersion vaccination of fish. In the present work, flounders (Paralichthys olivaceus) were immersed in three hyperosmotic solutions with 40, 50 and 60‰ salinities, then transferred into seawater of normal salinity (i.e. 30‰) containing formalin-inactivated Edwardsiella tarda for 30min. The antigen uptake in vaccinated flounder was determined using an absolute quantitative PCR (qPCR). The results showed significantly higher antigen uptake in the tissues of flounders immersed in solutions with 50‰ and 60‰ salinity compared to the control group directly immersed in vaccine (DI) (P<0.05), and the highest amount of antigen was detected in flounders immersed in the 50‰ salinity solution, whereas there was no significant difference in antigen uptake between the 40‰ salinity group and the DI group (P>0.05). A rapid and significant increase in antigen uptake was detected in the mucosal-associated tissues including the gill, skin and intestine (P<0.05) compared with the spleen, kidney and liver. Antigen uptake in the gill and skin both peaked at 30min post immersion, which was significantly higher than the levels of uptake measured in the other tissues (P<0.05), and then quickly declined. In contrast, antigen uptake in the spleen, kidney and liver gradually increased 3h post immersion (hpi). The expression profiles of four antigen presentation-related immune genes (MHC Iα, MHC IIα, CD4-1 and CD8α) were investigated after immersion. These four genes showed a significantly stronger response in the immersed flounders exposed to 50‰ salinity compared with the DI group (P<0.05). In the mucosal-associated tissues, the expression of MHC Iα and CD8α genes peaked at 24 hpi, while the expression of MHC IIα and CD4-1 genes showed up-regulation in the gill and skin and reached the peak in these tissues at 48 hpi. The expressions of the four genes were also up-regulated in spleen, kidney and liver, but reached peak expression in these tissues at 48-72 hpi. The results demonstrated that hyperosmotic immersion, notably 50‰ salinity significantly enhanced antigen uptake and the expression of selected genes associated with antigen presentation, providing evidence for an enhanced immune activation of the fish's immune response by the hyperosmotic immersion treatment prior to vaccination.

Dietary vitamin C reduced mercury contents in the tissues of juvenile olive flounder (Paralichthys olivaceus) exposed with and without mercury.

A 2×3 factorial design was employed to evaluate the effects of dietary vitamin C (l-ascorblyl-2-monophosphate, C2MP) levels on growth and tissue mercury (Hg) accumulations in juvenile olive flounder, Paralichthys olivaceus. Six experimental diets with two levels of mercuric chloride (0 or 20mg HgCl2/kg diet) and three levels of vitamin C (0, 100, or 200mg C2MP/kg diet) were added to the basal diet. At the end of 6 weeks feeding trial, in presence or absence of dietary Hg, fish body weight gain, specific growth rate, feed efficiency, protein efficiency ratio and whole body lipid content were increased in a dose-dependent manner as dietary vitamin C level increased in the diets. Interestingly, fish fed 100 or 200mg C2MP/kg diets showed significant interactive effects on reducing Hg content in kidney tissue. These results revealed that dietary vitamin C as 100 or 200mg C2MP/kg diet had protective effect against Hg accumulation in juvenile olive flounder.

Cloning and characterization of apoptosis-associated speck-like protein containing a CARD domain (ASC) gene from Japanese flounder Paralichthys olivaceus.

Apoptosis-associated speck-like protein containing a CARD domain (ASC) is a critical adaptor molecule in multiple inflammasome protein complexes that mediate inflammation and host defense. However, few studies have been performed in lower vertebrates such as in teleost. Here we identified and characterized a novel ASC gene (namely PoASC) from Japanese flounder Paralichthys olivaceus. The complete cDNA sequence of PoASC contains a 22bp 5'-untranslated sequence, a 612bp open reading frame, and a 438bp 3'-untranslated sequence. The deduced PoASC protein is comprised of 203 amino acids with a conserved N-terminal PYD domain and a C-terminal CARD domain and shows 35-62% sequence identity with other vertebrate ASC proteins. PoASC mRNA transcripts was detected in various Japanese flounder tissues and is dominantly expressed in hepatopancreas. Oligomeric speck-like structures were observed when PoASC was exogenously expressed in Japanese flounder FG-9307cells. Immune challenge experiments revealed that PoASC gene expression was significantly induced in the Japanese flounder head kidney macrophages and peripheral blood leukocytes by the canonical TLR ligands LPS, Poly(I:C) and zymosan stimulations. In addition, the induction of PoASC was also observed in Edwardsiella tarda challenged head kidney and gill tissues. Furthermore, we for the first time showed that extracellular ATP, an important signaling molecule in triggering innate immune response and activation of NLR inflammasome, significantly up-regulates PoASC expression in the Japanese flounder head kidney macrophages in a dose-dependent manner. Together, these findings addressed the involvement of PoASC in TLR and extracellular ATP-mediated innate immune signaling in the Japanese flounders.

Lipid metabolism during WSSV infection in shrimp

Dual-specificity MAP kinase (MAPK) phosphatases (DUSPs) are well-established negative modulators in regulating MAPK signaling in mammalian cells and tissues. Our previous studies have shown the involvement of DUSP6 in regulating innate immunity in Japanese flounder Paralichthys olivaceus. In order to gain a better understanding of the role of DUSPs in fish innate immunity, in the present study we identified and characterized three additional DUSP genes including DUSP1, 2 and 5 in P. olivaceus. The three Japanese flounder DUSP proteins share common domain structures composed of a conserved N-terminal Rhodanase/CDC25 domain and a C-terminal catalytic phosphatase domain, while they show only less than 26% sequence identities, indicating that they may have different substrate selectivity. In addition, mRNA transcripts of all the three DUSP genes are detected in all examined Japanese flounder tissues; however, DUSP1 is dominantly expressed in spleen while DUSP2 and 5 are primarily expressed in skin. Furthermore, all the three DUSP genes are constitutively expressed in the Japanese flounder head kidney macrophages (HKMs) and peripheral blood leucocytes (PBLs) with unequal distribution patterns. Moreover, all the three DUSPs gene expression was induced differently in response to the LPS and double-stranded RNA mimic poly(I:C) stimulations both in the Japanese flounder HKMs and PBLs, suggesting an association of DUSPs with TLR signaling in fish. Taken together, the co-expression of various DUSPs members together with their different responses to the immune challenges indicate that the DUSP members may operate coordinately in regulating the MAPK-dependent immune responses in the Japanese flounder.

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.

Lateral flow immunoassay for analyzing sulfadimethoxine residues in muscle tissue of olive flounder (Paralichthys olivaceus)

Antibiotic Detection Kit (Combination I), a lateral flow immunoassay (LFIA) developed for detecting antibiotic residues in milk, was utilized for the analysis of antibiotic residues in the muscle tissue of olive flounder. After 5-h treatment of samples by placing them in water dosed with sulfadimethoxine (SDM; 200 g/ton water), the residue depletion of SDM was investigated in 25 cultured olive flounders (Paralichthys olivaceus). Muscles from fish were sampled before treatment and on the 1st, 2nd, 3rd, 4th and 5th days after treatment. The concentration of SDM in the muscle was then determined by LFIA. The absorbance ratio of the sample to the control blank (Bs/Bo) was employed as an index to determine the residue in olive flounder muscle. To investigate the recovery rate, standard solutions were added to muscle samples to obtain final concentrations of 25 and 50 ng/mL in the muscle. The recovery rates of all spiked samples were >96.6% of the spiked value. SDM was detectable in the muscle of fish treated with the drug until the 1st day of the withdrawal period. The present study shows that the LFIA can be easily adopted to detect SDM residues in the tissue of farmed fish.

MiR-17 is involved in Japanese Flounder (Paralichthys olivaceus) development by targeting the Cdc42 mRNA.

The expression patterns of 197 miRNAs during Japanese flounder metamorphic development were recently analyzed. miR-17 was differentially expressed during the metamorphic period of the Japanese flounder; however, the role of miR-17 in Japanese flounder development has remained elusive to date. Bioinformatics analysis showed that Cdc42 was a putative target of miR-17. Cdc42 is a gene related to cell adhesion, migration, polarity, cytokinesis, growth, actin cytoskeleton, microtubule dynamics and transcription factor activity; thus, Cdc42 may contribute to metamorphic development. In our study, overexpression of miR-17 in FEC cells suppressed Cdc42 expression. The luciferase reporter assay confirmed that Cdc42 was the target of miR-17. The Cdc42 cDNA from the Japanese flounder was cloned and characterized for the first time. The expression of miR-17 was found to be negatively correlated with Cdc42 mRNA expression during temporal development and in the tissues of adult Japanese flounders. These results indicated that the decrease in miR-17 contributed to the up-regulation of Cdc42 during Japanese flounder metamorphosis. Cdc42 gene expression was down-regulated by thyroid hormone during Japanese flounder metamorphosis, whereas miR-17 was significantly up-regulated by thyroid hormone during these stages. These results indicated that miR-17 was a negative regulator of Cdc42.

Tissue Distribution of the 27.8 kDa Receptor and its Dynamic Expression in Response to Lymphocystis Disease Virus Infection in Flounder (Paralichthys olivaceus)

Lymphocystis disease virus (LCDV) enters and infects the gill cells of flounder (Paralichthys olivaceus) via a 27.8kDa membrane protein receptor. In the present study, immunohistochemistry was performed to locate the tissue distribution of this molecule in healthy flounder and showed that it was widely distributed in the tissues tested. Indirect enzyme-linked immunosorbent assay (ELISA) showed that the expression of the receptor in healthy flounder was highest in the gills and stomach, then in the skin, intestine and liver, followed by the spleen, head kidney, heart, ovary and brainand finally the kidney. On LCDV infection, ELISA indicated that the expression of the receptor, as determined by ELISA, was significantly upregulated in all tissues of LCDV-infected flounder compared with controls, but this expression decreased over the 4 weeks post infection. In contrast, real-time quantitative polymerase chain reaction demonstrated that the copy number of the LCDV gene in the tissues increased with time post infection, and that viral loads were higher in the tissues with higher expressions of the receptor. These results point to a correlation between high expression of the 27.8kDa receptor and efficient LCDV propagation. The wide tissue distribution of the receptor might be one reason why LCDV can infect various tissues leading to systemic infection.