Japanese Flounder Paralichthys Olivaceus Research Articles

Synchronously Mature Intersex Japanese Flounder (Paralichthys olivaceus): A Rare Case.

Japanese flounder is usually gonochoristic, with gonads that are either testes or ovaries. Here, we report an unusual case of hermaphroditism in Japanese flounder captured from the Bohai Sea. In the intersex flounder, the membrane of the upper ovary was closely connected to the abdominal muscles and internal organs, and the eggs filled the entire abdomen. The lower ovary was small and closely connected to the testes. The testes contained few fully mature sperm. Both eggs and sperm were capable of fertilization. The levels of several reproduction-related hormones (17β-estradiol, 11-ketotestosterone, 17α, 20β-dihydroxyprogesterone, luteinizing hormone, follicle-stimulating hormone, and testosterone) in the intersex flounder were intermediate, between those in females and males. The results showed that the heterozygosity of the intersex flounder was 0.632, and there were 28 single-nucleotide polymorphisms in the cyp21a gene. Compared with that of wild flounder, the activity of 21-hydroxylase was reduced by approximately 20.0%, and expressions of cyp19a, amh, and dmrt1 differed. We present the first report of its kind, detailing the anatomy, hormonal endocrinology, molecular biology, and physiology of the intersex Japanese flounder.

Identification and Functional Analysis of Ras-Related Associated with Diabetes Gene (rrad) in Edwardsiella piscicida-Resistant Individuals of Japanese Flounder (Paralichthys olivaceus).

Ras-related associated with diabetes (RRAD) is a member of the Ras GTPase superfamily that plays a role in several cellular functions, such as cell proliferation and differentiation. In particular, the superfamily acts as an NF-κB signaling pathway inhibitor and calcium regulator to participate in the immune response pathway. A recent transcriptome study revealed that rrad was expressed in the spleen of disease-resistant Japanese flounder (Paralichthys olivaceus) individuals compared with disease-susceptible individuals, and the results were also verified by qPCR. Thus, the present study aimed to explore how rrad regulates antimicrobial immunity via the NF-κB pathway. First, the coding sequence of P. olivaceus rrad was identified. The sequence was 1092 bp in length, encoding 364 amino acids. Based on phylogenetic and structural relationship analyses, P. olivaceus rrad appeared to be more closely related to teleosts. Next, rrad expression differences between disease-resistant and disease-susceptible individuals in immune-related tissues were evaluated, and the results revealed that rrad was expressed preferentially in the spleen of disease-resistant individuals. In response to Edwardsiella piscicida infection, rrad expression in the spleen changed. In vitro, co-culture was carried out to assess the hypo-methylated levels of the rrad promoter in the disease-resistant spleen, which was consistent with the high mRNA expression. The siRNA-mediated knockdown of rrad performed with the gill cell line of P. olivaceus affected many rrad-network-related genes, i.e., dcp1b, amagt, rus1, rapgef1, ralbp1, plce1, rasal1, nckipsd, prkab2, cytbc-1, sh3, and others, as well as some inflammation-related genes, such as bal2 and Il-1β. In addition, flow cytometry analysis showed that rrad overexpression was more likely to induce cell apoptosis, with establishing a link between rrad's function and its potential roles in regulating the NF-κB pathway. Thus,. the current study provided some clarity in terms of understanding the immune response about rrad gene differences between disease-resistant and disease-susceptible P. olivaceus individuals. This study provides a molecular basis for fish rrad gene functional analysis and may serve as a reference for in-depth of bacterial disease resistance of teleost.

Identification, expression, and function analysis of Rbpms2 splicing variants in Japanese flounder gonad

Rbpms2, an RNA-binding protein with multiple splicing (Rbpms), can interact with RNAs to involve oocyte development, thereby influencing female sex differentiation in vertebrates. Here, two splicing variants of the Rbpms2 gene from Japanese flounder (Paralichthys olivaceus) were identified, namely Rbpms2.1 and Rbpms2.2. The two variants exhibited 98.22 % amino acid homology, both featuring an RNA recognition motif (RRM) domain spanning positions 98–170 amino acids. They were relatively conserved throughout phylogenetic evolution. Differently, the C-terminal region of the Rbpms2.1 contains five additional sequential amino acids (–VRDQP–) compared to Rbpms2.2. The real-time qPCR results demonstrated that Rbpms2.1 and Rbpms2.2 had relatively abundant expression in the gonads of adult Japanese flounder, with higher expression levels in the ovary compared to the testis (P < 0.05). In situ hybridization results showed strong positive expression of Rbpms2 mRNA in oocytes at stages I-III during the V stage of ovarian development. In the testis atstage IV, the expression of Rbpms2 mRNA was mainly concentrated on primary spermatocytes. Importantly, Rbpms2 binding sites were found in the 3′UTR, 5′UTR, and ORF regions of the sex-related genes including dmrt1, sox9, amh, foxl2, and wnt4. siRNA interference and overexpression analysis of Rbpms2.1 and Rbpms2.2 in primary cells of the ovary and testis showed that Rbpms2 can repress the expression of male-related genes (dmrt1, sox9, and amh) and significantly promote the expression of female-related genes (foxl2 and wnt4). Our results revealed that Rbpms2 may play a critical role by targeting the sex-related genes in the gonad development of Japanese flounder.

PoIL8-L, a teleost interleukin-8 like, enhances leukocyte cellular vitality and host defense against bacterial infections in Japanese flounder (Paralichthys olivaceus)

Interleukin-8 (IL-8), a CXC chemokine, exerts pivotal effect on cell migration, inflammatory response, and immune regulation. In this study, we examined the immunological characteristics of an IL-8 like homologue (PoIL8-L) in Japanese flounder (Paralichthys olivaceus). PoIL8-L contains a conserved chemokine CXC domain and 105 amino acid residues. PoIL8-L expression in tissues was constitutive, and significantly regulated by V. havieri or E. tarda infection. In vitro, rPoIL8-L could bind to eight tested bacteria, exhibited bacteriostatic and bactericidal effects against certain bacteria, and could bind to the targeted bacterial Ⅳ pilin protein rPilA of E. tarda. Furthermore, rPoIL8-L could attach to peripheral blood leukocytes, and enhance their immune genes expression, respiratory burst, chemotaxis, proliferation, acid phosphatase activity, and phagocytic activity. Additionally, rPoIL8-L induce neutrophils to extrude neutrophil extracellular traps. In vivo, rPoIL8-L could promote host resistance to E. tarda infection. In summary, these findings provide fresh perspectives on the immunological antibacterial properties of IL-8 in teleost.

Effect of heat exposure on histology, transcriptomics and co-expression network: A synthetic study in Japanese flounder (Paralichthys olivaceus)

Water temperature has continued to increase as part of global warming. The heart of fish is the center that participates in the physiological limitations of thermal performance. However, only a few studies have focused on the response mechanisms of fish hearts to heat stress. Therefore, the economically important cold-water aquaculture fish, Japanese flounder (Paralichthys olivaceus) was chosen to explore the mechanisms of the heart in response to heat stress. Histological observations indicated that heat stress could widen the sarcoplasmic distance and deepen chromatin staining. Further transcriptome analysis identified 958 differentially expressed genes (DEGs), and significantly enriched “Protein processing in endoplasmic reticulum” pathway and related terms in functional enrichment analysis. Weighted gene co-expression network analysis (WGCNA) showed that different tissues had different response modules and hub genes, and calcium ions (Ca2+) related genes and pathways were noteworthy in the heart specific module. Functional identification of key Ca2+ regulatory hub genes illuminated that caveolin-3 (cav3) was an important gene that participates in controlling the Ca2+ into and out of cells. Meanwhile, overexpression of cav3 could reduce heat stress induced apoptosis. This study could not only be a crucial resource for the response mechanisms underlying heat stress in Japanese flounder, but also provide valuable insights into genetic improvement for heat-resistance.

Genome-wide association study for high-temperature tolerance in the Japanese flounder

This study addresses the critical issue of high-temperature stress in Japanese flounder (Paralichthys olivaceus), a factor threatening both their survival and the growth of the aquaculture industry. The research aims to identify genetic markers associated with high-temperature tolerance, unravel the genetic regulatory mechanisms, and lay the foundation for breeding Japanese flounder with increased resistance to high temperatures. In this study, using a genome-wide association study was performed to identify single nucleotide polymorphisms (SNPs) and genes associated with high−temperature tolerance for Japanese flounder using 280 individuals with 342 311 high-quality SNPs. The traits of high-temperature tolerance were defined as the survival time and survival status of Japanese flounder at high water temperature (31℃) for 15 days cultivate. A genome-wide association study identified six loci on six chromosomes significantly correlated with survival time under high-temperature stress. Six candidate genes were successfully annotated. Additionally, 34 loci associated with survival status were identified and mapped to 15 chromosomes, with 22 candidate genes annotated. Functional analysis highlighted the potential importance of genes like traf4 and ppm1l in regulating apoptosis, impacting high-temperature tolerance in Japanese flounder. These findings provide a valuable theoretical framework for integrating molecular markers into Japanese flounder breeding programmes, serving as a molecular tool to enhance genetic traits linked to high-temperature tolerance in cultured Japanese flounder.

Aconitate decarboxylase 1 (Acod1) suppresses inflammatory stimuli-induced immune responses in Japanese flounder (Paralichthys olivaceus)

Aconitate decarboxylase 1 (ACOD1, formerly termed as immunoresponsive gene 1) is an enzyme responsible for generation of anti-inflammatory metabolite itaconate through decarboxylation of cis-aconitate and plays a key role in inflammation regulation and anti-microbial host defense in mammals. However, the immune functions of Acod1 in fish remain largely undetermined. Here, we characterize a Acod1 homolog that is highly induced by LPS, poly(I:C) and extracellular ATP (eATP) challenges in the Japanese flounder Paralichthys olivaceus head kidney macrophages (HKMs). We find that Acod1 syntenic genes in P. olivaceus are similar with those in mammalian species and the deduced Japanese flounder ACOD1 protein shares similar protein domains and 3-D structures with its counterparts in mammals. We next show that itaconate derivatives dimethyl itaconate (DMI) and citraconate (Cit) can markedly down-regulate inflammatory stimuli, including LPS-, poly(I:C)- and eATP-induced inflammatory mediator nitric oxide (NO) production and gene expression of proinflammatory cytokines (IL-1β and TNF-α), chemokines (CXCL-8, CXCL-9 and CXCL-13), HMGB1 and inflammasome components (NLRP3, NLRC3, caspase1, ASC and GSDME), but up-regulate anti-inflammatory gene (IL-10 and SOCS1/3) expression in P. olivaceus HKMs. In addition, DMI and Cit treatment significantly inhibits Edwardsiella piscicida growth in P. olivaceus HKMs. Furthermore, knockdown of the endogenous Acod1 expression by siRNA silencing not only significantly increases LPS-, poly(I:C)- and eATP-induced proinflammatory gene expression but also decreases anti-inflammatory gene expression in the HKMs. In contrast, the LPS-, poly(I:C)- and eATP-indued proinflammatory gene expression and NO production are greatly attenuated in ACOD1 overexpressed Japanese flounder FG-9307 cells. Collectively, our studies demonstrate that the inducible fish Acod1 gene is a novel immune regulator in dampening inflammatory stimuli-induced immune responses and ACOD1-produced itaconate may play an essential role in anti-bacterial immunity in fish macrophages.

A fungal-binding agglutinin in the skin slime of Japanese flounder (Paralichthys olivaceus) is glyceraldehyde 3-phosphate dehydrogenase.

Agglutination of pathogenic microorganisms on the body surface is a significant phenomenon for the prevention of infection. In the present study, we show that an extract of the skin mucus from Japanese flounder (Paralichthys olivaceus) has agglutination activity against the yeast Saccharomyces cerevisiae. We purified this yeast-binding protein, which consists of an approximately 35-kDa homodimer, using affinity chromatography with yeast as a ligand. Multiple internal amino acid sequences of the protein, as determined using liquid chromatography with quadrupole time-of-flight tandem mass spectrometry, mapped to flounder glyceraldehyde 3-phosphate dehydrogenase (GAPDH). An anti-GAPDH antibody inhibited the yeast agglutination activity in the skin mucus extract and stained agglutinated yeast, indicating that flounder GAPDH could agglutinate yeast. The current study suggests that GAPDH, a well-known protein as the sixth enzyme in the glycolytic pathway, is a significant player in mucosal immunity in teleosts.

Effects of inactivated Streptococcus iniae, Edwardsiella tarda, and Poly I:C on mRNA Expression Levels of CXCL-10 and CXCL-9 Genes in Japanese Flounder

The present study accomplished the successful cloning and sequencing of the JfCXCL9_L and JfCXCL10_L genes found in the spleen cDNA of the Japanese flounder (Paralichthys olivaceus). The tissue distribution of these two genes was determined before any stimuli administration at the zero-hour mark. The qRT-PCR analysis demonstrated that the expression of JfCXCL10_L closely mirrored that of IL1-β, displaying an upregulation following the application of Poly I:C (Viral mimic) and formalin-killed Edwardsiella tarda (Gram-Negative mimic), while showing a downregulation after the application of formalin-killed Streptococcus iniae (Gram-Positive mimic) treatment. These findings strongly suggest a role for JfCXCL10_L in the immune response to viral and gram-negative bacterial stimuli. Regarding JfCXCL9_L, mRNA levels were found to be significantly downregulated after FKCET and FKCSI treatments, though to varying extents. Interestingly, at specific time points, JfCXCL9_L levels were even lower compared to Poly I:C treatment. These intriguing findings shed valuable light on the roles of both JfCXCL9_L and JfCXCL10_L in potential functions of immune response mechanisms of the Japanese flounder'.

DNA Methylation Mediates Sperm Quality via piwil1 and piwil2 Regulation in Japanese Flounder (Paralichthys olivaceus).

DNA methylation is an important way to regulate gene expression in eukaryotes. In order to reveal the role of DNA methylation in the regulation of germ cell-specific piwi gene expression during spermatogenesis of Japanese flounder (Paralichthys olivaceus), the expression profiles of piwil1 (piwi-like 1) and piwil2 (piwi-like 2) genes in the gonads of female, male, and sex-reversed pseudo-male P. olivaceus were analyzed, and the dynamic of DNA methylation was investigated. As a result, piwil1 and piwil2 genes were highly expressed in the testis of both male and pseudo-male P. olivaceus, with significant variation among male individuals. The DNA methylation levels in the promoter regions of both piwil1 and piwil2 were negatively correlated with their expression levels, which may contribute to the transcriptional regulation of piwi genes during spermatogenesis. There was also sperm quality variation among male P. olivaceus, and the sperm curvilinear velocity was positively correlated with the expression of both piwil1 and piwil2 genes. These results indicated that the DNA methylation in piwil1 and piwil2 promoter regions may affect the initiation of piwi gene transcription, thereby regulating gene expression and further affecting the spermatogenesis process and gamete quality in P. olivaceus.

Coordinated regulatory feedback between estradiol and miR-17-92 cluster in ovaries of Japanese flounder Paralichthys olivaceus

Endogenous microRNAs (miRNAs), often distributed in clusters, can enable the subtle and precise regulation in gene expression via a variety of ways and different regions. In this study, multiple regulatory patterns for the estrogenic cyp19a1a and hsd17b12a genes by miR-17-92 cluster (miR-17, miR-18a, miR-19a, miR-20a, miR-19b and miR-92a) were investigated in ovaries of Japanese flounder (Paralichthys olivaceus), the important marine cultured fish in North Asia with sexual dimorphism in growth and sex reversal scenario. Through dual-luciferase reporter assay, the regulation of P. olivaceus estrogenic genes by miR-17-92 cluster could be the combined multi-effect between 3′-UTR and 5′-ends targeting. For cyp19a1a, miR-17/20a induced its expression by binding to the promoter but simultaneously inhibited cyp19a1a through 3′-UTR targeting, while miR-92a activated cyp19a1a by binding to the promoter, which made the overall up-regulation of cyp19a1a more significant. For hsd17b12a, the binding of miR-17/20a and miR-92a to the promoter and the binding of miR-18a to 3′-UTR all inhibited its expression, which ultimately reduced the hsd17b12a level. Both in vitro and in vivo experiments in P. olivaceus ovaries confirmed these canonical and non-canonical regulatory patterns of miR-17-92 cluster, which coordinately reduced the estradiol (E2) level. Moreover, E2 could act as a repressor of miR-17-92 cluster, which may form a regulatory feedback loop whereby E2 regulates estrogenic process through miR-17-92 cluster. These results contributed to a deeper understanding of the miR-17-92 cluster function in teleosts and provided more insights into the molecular mechanism of fish reproductive regulation.

Consumer preference for label presentations of freshness, taste, and serving suggestion on fresh fish packages of Japanese flounder Paralichthys olivaceus

Consumer preference for label presentations of freshness, taste, and serving suggestion on fresh fish packages of Japanese flounder Paralichthys olivaceus

Age, growth, and estimation of the age-length key for Japanese flounder Paralichthys olivaceus in the southwestern Sea of Japan

Age, growth, and estimation of the age-length key for Japanese flounder Paralichthys olivaceus in the southwestern Sea of Japan

A single C1q A chain promotes leukocytes phagocytosis/chemotaxis and mediates bacterial clearance in Japanese flounder Paralichthys olivaceus

Complement component 1q (C1q) is the first component involved in the activation of complement classical pathway and acts as a linker of the innate and adaptive immune system. The C1q is a macromolecular containing three chains i.e. C1q A, C1q B, and C1q C. In teleost, the biological functions of C1q A chain is still unclear. In this study, we investigated the immunological properties of a C1q A chain from Japanese flounder (Paralichthys olivaceus) (PoC1qA). PoC1qA containing the conserved CLR and C1q domain, shares 45.4%–78.6% identities with that of other teleost and mammals. PoC1qA expression pattern was constitutive in tissues, with higher expression in spleen and liver. Besides, the PoC1qA expression was notably regulated by pathogenic invasion. In vitro the recombinant PoC1qA (rPoC1qA) could directly bind a variety of bacteria, and affect bacterial growth and survival. Moreover, PoC1qA could influence the hemolysis and bactericidal activity of fish serum. Additionally, rPoC1qA could interact with the membrane of peripheral blood leucocytes (PBLs), promoting the PBLs phagocytosis and chemotaxis, and enhancing the activation of PBLs. Consistently, in vivo rPoC1qA could restrain the bacterial replication in immune tissues. To sum up, our observations added the first evidence that an A chain of teleost C1q plays vital roles in mediating PBLs activity and antibacterial immunity.

Identification and functional analysis of circpdlim5a generated from pdlim5a gene splicing in the skeletal muscle of Japanese flounder (Paralichthys olivaceus)

Circular RNAs (circRNAs) are non-coding RNAs with endogenous regulatory functions, including regulating skeletal muscle development. However, its role in the development of skeletal muscle in Japanese flounder (Paralichthys olivaceus) is not clear. Therefore we screened a candidate circpdlim5a, which is derived from the gene pdlim5a, from the skeletal muscle transcriptome of Japanese flounder. We characterized circpdlim5a, which was more stable compared to the linear RNA pdlim5a. Distributional characterization of circpdlim5a showed that circpdlim5a was predominantly distributed in the nucleus and was highly expressed in the skeletal muscle of adult Japanese flounder (24 months). When we further studied the circpdlim5a function, we found that it inhibited the expression of proliferation and differentiation genes according to the over-expression experiment of circpdlim5a in myoblasts. We concluded that circpdlim5a may inhibit the proliferation and differentiation of myoblasts and thereby inhibit skeletal muscle development in Japanese flounder. This experiment provides information for the study of circRNAs by identifying circpdlim5a and exploring its function, and offers clues for molecular breeding from an epigenetic perspective.

Post-settlement dynamics of Japanese flounder Paralichthys olivaceus in Tango Bay: Seasonal patterns in growth, mortality, and recruitment potential.

Japanese flounder Paralichthys olivaceus is one of the most valuable coastal flatfish species in East Asia. To investigate post-settlement growth and mortality, juveniles were sampled in Tango Bay (Japan) weekly throughout the settlement period in 2007 and 2008. Otolith (lapillus) microstructure analysis enabled the categorization of juveniles into six biweekly cohorts each year. Later cohorts exhibited higher growth rates possibly because of higher water temperatures. A key observation was the direct relationship between high mortality and high density in mid-season cohorts in both years, pointing to density-dependent mortality. This increased mortality may be attributed to predation, including cannibalism by earlier cohorts. Furthermore, growth-selective mortality was evident soon after settlement, underscoring the vulnerability of slow growers to predation during the early juvenile stage. Although earlier and later cohorts were less abundant but showed promising recruitment potential, the prospective contribution of mid-season cohorts to the adult population remained uncertain. The results clearly highlight the importance of density-dependent mortality in population regulation in post-settlement Japanese flounder.

Functional analysis of the ube3a response in Japanese flounder (Paralichthys olivaceus) to CSBV infection

Ube3a is a member of the E3 ubiquitin ligase HECTc family, and its role has been established in neurodevelopmental disorders. However, studies on its role in Japanese flounder are scarce. Thus, in this study, the ube3a of Japanese flounder was cloned, and its role in conferring resistance against Chinook salmon bafnivirus (CSBV) was analyzed. Japanese flounder ube3a encoded a protein containing 834 amino acids. Interestingly, its homology with the Atlantic halibut was determined to be 94%. In addition, there were differential expressions of ube3a in different tissues of Japanese flounder, with the highest expression level observed in the fin, followed by the gills and skin (P ≤ 0.05). Subcellular localization analysis revealed that Ube3a is a cytoplasmic protein. We established an in vitro CSBV infection model using Japanese flounder gill cell line (FG). After ube3a overexpression, the viral load was significantly lower than that of the control group (P ≤ 0.05). Contrastingly, after incubation of FG cells with an E3 ubiquitin ligase inhibitor, the viral load was significantly higher than in the control group (P ≤ 0.01). Then, the expression levels of nf-κb, traf3, and tnf-α after incubation with an E3 ubiquitin ligase inhibitor were examined. The results demonstrated that ube3a may exerted a significant antiviral effect in Japanese flounder via the ubiquitination pathway.

Intestinal epithelial cells of Japanese flounder (Paralichthys olivaceus) as an in vitro model for studying intestine immune function based on transcriptome analysis

Japanese flounder (Paralichthys olivaceus) is an economically crucial marine species, but diseases like hemorrhagic septicemia caused by Edwardsiella tarda have resulted in significant economic losses. E. tarda infects various hosts, and its pathogenicity in fish is not fully understood. Lipopolysaccharides (LPS) are components of the outer membrane of Gram-negative bacteria and are representative of typical PAMP molecules that cause activation of the immune system. The PoIEC cell line is a newly established intestinal epithelial cell line from P. olivaceus. In order to investigate whether it can be used as an in vitro model for studying the pathogenesis of E. tarda and LPS stimulation, we conducted RNA-seq experiments for the PoIECs model of E. tarda infection and LPS stimulation. In this study, transcriptome sequencing was carried out in the PoIEC cell line after treatment with LPS and E. tarda. A total of 62.52G of high-quality data from transcriptome sequencing results were obtained in nine libraries, of which an average of 87.96% data could be aligned to the P. olivaceus genome. Data analysis showed that 283 and 414 differentially expressed genes (DEGs) in the LPS versus Control (LPS-vs-Con) and E. tarda versus Control groups (Et-vs-Con), respectively, of which 60 DEGs were shared in two comparation groups. The GO terms were predominantly enriched in the extracellular space, inflammatory response, and cytokine activity in the LPS-vs-Con group, whereas GO terms were predominantly enriched in nucleus and positive regulation of transcription by RNA polymerase II in the Et-vs-Con group. KEGG analysis revealed that three immune-related pathways were co-enriched in both comparison groups, including the Toll-like receptor signaling pathway, C-type lectin receptor signaling pathway, and Cytokine-cytokine receptor interaction. Five genes were randomly screened to confirm the validity and accuracy of the transcriptome data. These results suggest that PoIEC cell line can be an ideal in vitro model for studies of marine fish gut immunity and pathogenesis of Edwardsiellosis.

LncRNA pol-lnc78 as a ceRNA regulates antibacterial responses via suppression of pol-miR-n199-3p-mediated SARM down-regulation in Paralichthys olivaceus.

Long non-coding RNAs (lncRNAs) function as key modulators in mammalian immunity, particularly due to their involvement in lncRNA-mediated competitive endogenous RNA (ceRNA) crosstalk. Despite their recognized significance in mammals, research on lncRNAs in lower vertebrates remains limited. In the present study, we characterized the first immune-related lncRNA (pol-lnc78) in the teleost Japanese flounder ( Paralichthys olivaceus). Results indicated that pol-lnc78 acted as a ceRNA for pol-miR-n199-3p to target the sterile alpha and armadillo motif-containing protein (SARM), the fifth discovered member of the Toll/interleukin 1 (IL-1) receptor (TIR) adaptor family. This ceRNA network regulated the antibacterial responses of flounder via the Toll-like receptor (TLR) signaling pathway. Specifically, SARM acted as a negative regulator and exacerbated bacterial infection by inhibiting the expression of inflammatory cytokines IL-1β and tumor necrosis factor-α (TNF-α). Pol-miR-n199-3p reduced SARM expression by specifically interacting with the 3' untranslated region (UTR), thereby promoting SARM-dependent inflammatory cytokine expression and protecting the host against bacterial dissemination. Furthermore, pol-lnc78 sponged pol-miR-n199-3p to ameliorate the inhibition of SARM expression. During infection, the negative regulators pol-lnc78 and SARM were significantly down-regulated, while pol-miR-n199-3p was significantly up-regulated, thus favoring host antibacterial defense. These findings provide novel insights into the mechanisms underlying fish immunity and open new horizons to better understand ceRNA crosstalk in lower vertebrates.

Role of Thyroid Hormone in Dynamic Variation of gdf6a Gene during Metamorphosis of Paralichthys olivaceus.

The Japanese flounder (Paralichthys olivaceus) is a marine fish that undergoes a dramatic postembryonic metamorphosis, with the right eye shifting to the left and its lifestyle transitioning from planktonic to benthic. As the light environment of the habitat changes from bright to dim, its photoreceptor system also undergoes adaptive change. Growth differentiation factor 6a (Gdf6a) is a member of the BMP family, which plays a key role in regulating the dorsal-ventral pattern of the retina and photoreceptor fate, and the differentiation of different photoreceptors is also modulated by a thyroid hormone (TH) binding its receptor (TR). However, the relationship between gdf6a and TH and its role in the regulation of photoreceptors during flounder metamorphosis is still poorly understood. In this study, bioinformatics analysis showed that Gdf6a had a conserved TGFB structural domain and clusters with fishes. The expression analysis showed that the expression of gdf6a was highest in the eye tissue of adult flounder and tended to increase and then decrease during metamorphosis, reaching its highest levels at the peak of metamorphosis. Moreover, the expression of gdf6a increased in the early stages of metamorphosis after exogenous TH treatment, while it was inhibited after exogenous thiourea (a TH inhibitor, TU) treatment. To further investigate the targeting role of TH and gdf6a in the metamorphosis of flounder, the results of the Dual-Luciferase revealed that triiodothyronine (T3) may regulate the expression of gdf6a through TRβ. In conclusion, we speculate that TH influences the development of cone photoreceptors during the metamorphosis of the flounder by regulating the expression of gdf6a.