Molecular characterization and ontogenetic expression profiles of LPXRFa and its receptor in Japanese flounder (Paralichthys olivaceus).

Investigations concerning the LPXRFa system are rarely conducted in flatfish species. Here, we first identified and characterized lpxrfa and its cognate receptor lpxrfa-r genes in the Japanese flounder (Paralichthys olivaceus). The coding DNA sequence of lpxrfa was 579 bp in length, wich encoded a 192-aa preprohormone that can produce three mature LPXRFa peptides. The open reading frame (ORF) of lpxrfa-r was 1446 bp in size, and encoded a 481-aa LPXRFa-R protein that encompassed seven hydrophobic transmembrane domains. Subsequently, tissue distribution expression profiles of lpxrfa and lpxrfa-r transcripts were assayed by quantitative real-time PCR. The results indicated that expressions of lpxrfa transcripts were detected at the highest levels in the brain of both females and males, however, lpxrfa-r transcripts were remarkablely expressed in the brain tissue of female fish and in the testis tissue of male fish. Furthermore, transcript levels of lpxrfa and lpxrfa-r genes were investigated during early ontogenetic development, with the maximum expression levels at 30 days post-hatching. Overall, these data contribute to providing preliminary proof for the existence and structure of the LPXRFa system in Japanese flounder, and the study is just the foundation for researching physiological function of LPXRFa system in this species.

Differences on the level of hepatic transcriptome between two flatfish species in response to liver cancer and environmental pollution levels.

Environmental factors can cause cancer in both wild animals and humans. In ecological settings, genetic variation and natural selection can sometimes produce resilience to the negative impacts of environmental change. An increase in oncogenic substances in natural habitats has therefore, unintentionally, created opportunities for using polluted habitats to study cancer defence mechanisms. The Baltic and North Sea are among the most contaminated marine areas, with a long history of pollution. Two flatfish species (flounder, Platichthys flesus and dab, Limanda limanda) are used as ecotoxicological indicator species due to pollution-induced liver cancer. Cancer is more prevalent in dab, suggesting species-specific differences in vulnerability and/or defence mechanisms. We conducted gene expression analyses for 30 flatfishes. We characterize between- and within-species patterns in potential cancer-related mechanisms. By comparing cancerous and healthy fishes, and non-cancerous fishes from clean and polluted sites, we suggest also genes and related physiological mechanisms that could contribute to a higher resistance to pollution-induced cancer in flounders. We discovered changes in transcriptome related to elevated pollutant metabolism, alongside greater tumour suppression mechanisms in the liver tissue of flounders compared to dabs. This suggests either hormetic upregulation of tumour suppression or a stronger natural selection pressure for higher cancer resistance for flounders in polluted environment. Based on gene expression patterns seen in cancerous and healthy fish, for liver cancer to develop in flounders, genetic defence mechanisms need to be suppressed, while in dabs, analogous process is weak or absent. We conclude that wild species could offer novel insights and ideas for understanding the nature and evolution of natural cancer defence mechanisms.

Two novel teleost calreticulins PoCrt-1/2, with bacterial binding and agglutination activity, are involved in antibacterial immunity.

Calreticulin (Crt), a conserved lectin-like pleiotropic protein, plays crucial roles in mammalian immune response. In fish, the immunological function of Crt is limited investigated. Herein, we studied the antibacterial immunity of two type of Crt homologues (i.e. PoCrt-1 and PoCrt-2) in Japanese flounder (Paralichthys olivaceus). PoCrt-1 and PoCrt-2 are composed of 419 and 427 amino acid residues respectively, with 69.09% overall sequence identities with each other. Both PoCrt-1 and PoCrt-2 contain a signal peptide and three functional domains i.e. N-, P- and C-domains. Both PoCrt-1 and PoCrt-2 were constitutively expressed at various tissues with highest expression level in liver, and obviously regulated by Edwardsiella tarda and Vibrio harveyi. Furthermore, recombinant PoCrt-1 and PoCrt-2 (rPoCrt-1 and rPoCrt-2) could bind to different Gram-negative bacteria with highest binding index with E. tarda. At same time, in vitro rPoCrt-1 and rPoCrt-2 could agglutinate E. tarda, V. harveyi, and Vibrio anguillarum, and inhibit the bacterial growth. Similarly, in vivo rPoCrt-1 and rPoCrt-2 could significantly suppress the dissemination of E. tarda. Overall, these observations add new insights into the antibacterial immunity of Crt in P. olivaceus.

Toxic effects of combined exposure to cadmium and nitrate on intestinal morphology, immune response, and microbiota in juvenile Japanese flounder (Paralichthys olivaceus).

Cadmium (Cd2+) and nitrate (NO3-) are important environmental pollutants in the offshore marine ecological environment. However, limited research has explored their combined effects, particularly regarding their impact on the microbiota and intestinal health of marine fish. In this study, juvenile Japanese flounders (P. olivaceus) were immersed in seawater samples with different combinations of Cd2+ (0, 0.2, and 2 mg/L) and NO3- (0 and 80 mg/L NO3N) for 30 days to explore their toxic impacts on intestinal morphology, tight junction (TJ) barrier, immune response, and microbiota. Our results showed that Cd2+ or NO3- exposure alone led to histopathological damage of the gut, while their co-exposure aggravated intestinal damage. Moreover, co-exposure substantially decreased TJ-related gene expression, including occludin, claudin-10, and ZO-2, suggesting increased TJ permeability in the gut. Regarding the immune response, we observed upregulated expression of immune-related markers such as HSP40, IL-1ß, TNF-α, and MT, suggesting the onset of intestinal inflammation. Furthermore, Cd2+ and NO3- exposure led to changes in intestinal microflora, characterized by decreased the abundance of Sediminibacterium and NS3a_marine_group while increasing the prevalence of pathogens or opportunistic pathogens such as Ralstonia, Proteus, and Staphylococcus. This alteration in microbiota composition increased network complexity and α-diversity, ultimately causing dysbiosis in the fish gut. Additionally, combined exposure resulted in metabolic disorders that affected the predicted functions of the intestinal microbiota. Overall, our study demonstrates that Cd2+-NO3- co-exposure amplifies the deleterious effects compared to single exposure. These findings enhance our understanding of the ecological risks posed by Cd2+-NO3- co-exposure in marine ecosystems.

Genome-wide DNA methylation mediates the resistance to vibriosis in Cynoglossus semilaevis.

Chinese tongue sole (Cynoglossus semilaevis) is an economically important marine fish in China. However, vibriosis has caused huge mortality and economic losses in its culturing industry. To reveal the effect of DNA methylation on the resistance to vibriosis in tongue sole, we conducted RNA sequencing and whole genome bisulfite sequencing (WGBS), and compared the gene expressions and DNA methylation patterns between the resistant and susceptible families. We identified a total of 741 significantly differentially expressed genes (DEGs) in kidney and 17460 differentially methylated genes (DMGs), which were both enriched in immune-related pathways, such as "cAMP signaling pathway" and "NOD-like receptor signaling pathway". Through the correlation analysis of DEGs and DMGs, we identified two important immune pathways, including "complement and coagulation cascades", and "cytokine-cytokine receptor interaction", which played important roles in regulating the inflammation level and immune homeostasis. For example, the expression of proinflammatory cytokine il17c was down-regulated under the regulation of DNA methylation; in addition, the expression of protease-activated receptor 3 (par3) was up-regulated, which could induce the up-expressionof il8. These results demonstrated that the regulation of DNA methylation on the genes involved in immune responses might contribute to the resistance to vibriosis in tongue sole, and provided a basis for the control of diseases in fish aquaculture.

Formulation of chitosan microsphere-based oral vaccine against the scuticociliate parasite Miamiensis avidus: Evaluation of its protective immune potency in olive flounder (Paralichthys olivaceus).

Miamiensis avidus is a parasitic pathogen that causes scuticociliatosis, a severe and often lethal marine infection that affects marine fishes worldwide, including olive flounder (Paralichthys olivaceus) in Korea. This parasite infects all size groups of flounder year-round, causing recurring mortalities and huge economic losses to the Korean flounder industry each year. However, few efforts have been made to implement effective remedial measures to control this parasite. Therefore, our study sought to develop a chitosan microsphere (MS)-encapsulated inactivated vaccine (IMa + chitosan) for oral delivery (adsorbed in feed) to flounder fingerlings and assess its protective efficacy at different modalities via three in vivo experimental trials. Immunisation trial-1 was conducted to determine the effective concentration of chitosan. Our findings indicated that an IMa + chitosan 0.05 % vaccine formulation was safe and effective in providing moderate protection [46.67%-53.3 % relative percent survival (RPS)] against M. avidus intraperitoneal (IP) injection challenge at two weeks post-vaccination (wpv) compared to the IMa + chitosan 0.01 % and IMa + chitosan 0.005 % vaccines (0%-13.3 % RPS) irrespective of the antigen doses. In trial-2, the IMa + chitosan 0.05 % vaccine elicited similar protective immunity (30.8%-57.1 % RPS) in olive flounder against M. avidus at varying antigen doses (high: 2.38 × 106 cells/fish; low: 1.5 × 105 cells/fish), immunisation periods (2 and 5 wpv), and challenge modes (IP injection and immersion). Furthermore, experimental trial-3 validated the use of chitosan MS as an IMa antigen carrier to improve survivability (41.7 % RPS) in the host by significantly (p < 0.05) upregulating specific anti-M. avidus antibody titres in the fish sera and mucus of the group immunised with IMa-containing chitosan MS. In contrast, non-specific immunomodulatory effects (16.7 % RPS and enhanced mucosal antibody titres) were observed in the group treated with chitosan MS without IMa. Therefore, our findings suggested that oral administration of chitosan MS (0.05 %)-encapsulated IMa vaccine is a promising immunisation strategy against M. avidus that can protect the IMa antigen from digestive degradation, facilitates its targeted delivery to the host immune organs, and helps in orchestrating protective immune induction in olive flounder, thus controlling parasite infection.

Characterization and function of Japanese flounder (Paralichthys olivaceus) slc2a6 in response to lymphocystis disease virus infection.

Slc2a6 is a member of the slc2 family (solute carrier 2 family) and previous reports have indicated its involvement in the inflammatory response. Slc2a6 is regulated by the NF-ĸB signaling pathway. This study investigated the differential expression of slc2a6 in the early embryonic development of Japanese flounder, revealing that the early gastrula stage had the highest level of slc2a6 expression. Moreover, slc2a6 expression was increased in vitro after stimulation by lymphocystis disease virus (LCDV), and in vivo experiments also showed significantly elevated levels in the spleen and muscle tissues following LCDV stimulation. Subcellular localization revealed that Slc2a6 was expressed in both the nucleus and cytoplasm of cells. The pcDNA3.1-slc2a6 overexpression plasmid was successfully constructed; the si-slc2a6 interfering strand was screened and samples were collected. The expression of NF-ĸB signaling pathway-related genes il-1ß, il-6, nf-ĸb, and tnf-α was evaluated in overexpressed, silenced, and LCDV-stimulated samples. The results showed that slc2a6 is involved in viral regulation in Japanese flounder by regulating innate immune responses.

Transcriptome analysis reveals molecular mechanisms of lymphocystis formation caused by lymphocystis disease virus infection in flounder (Paralichthys olivaceus).

Lymphocystis disease is frequently prevalent and transmissible in various teleost species worldwide due to lymphocystis disease virus (LCDV) infection, causing unsightly growths of benign lymphocystis nodules in fish and resulting in huge economic losses to aquaculture industry. However, the molecular mechanism of lymphocystis formation is unclear. In this study, LCDV was firstly detected in naturally infected flounder (Paralichthys olivaceus) by PCR, histopathological, and immunological techniques. To further understand lymphocystis formation, transcriptome sequencing of skin nodule tissue was performed by using healthy flounder skin as a control. In total, RNA-seq produced 99.36%-99.71% clean reads of raw reads, of which 91.11%-92.89% reads were successfully matched to the flounder genome. The transcriptome data showed good reproducibility between samples, with 3781 up-regulated and 2280 down-regulated differentially expressed genes. GSEA analysis revealed activation of Wnt signaling pathway, Hedgehog signaling pathway, Cell cycle, and Basal cell carcinoma associated with nodule formation. These pathways were analyzed to interact with multiple viral infection and tumor formation pathways. Heat map and protein interaction analysis revealed that these pathways regulated the expression of cell cycle-related genes such as ccnd1 and ccnd2 through key genes including ctnnb1, lef1, tcf3, gli2, and gli3 to promote cell proliferation. Additionally, cGMP-PKG signaling pathway, Calcium signaling pathway, ECM-receptor interaction, and Cytokine-cytokine receptor interaction associated with nodule formation were significantly down-regulated. Among these pathways, tnfsf12, tnfrsf1a, and tnfrsf19, associated with pro-apoptosis, and vdac2, which promotes viral replication by inhibiting apoptosis, were significantly up-regulated. Visual analysis revealed significant down-regulation of cytc, which expresses the pro-apoptotic protein cytochrome C, as well as phb and phb2, which have anti-tumor activity, however, casp3 was significantly up-regulated. Moreover, bcl9, bcl11a, and bcl-xl, which promote cell proliferation and inhibit apoptosis, were significantly upregulated, as were fgfr1, fgfr2, and fgfr3, which are related to tumor formation. Furthermore, RNA-seq data were validated by qRT-PCR, and LCDV copy numbers and expression patterns of focused genes in various tissues were also investigated. These results clarified the pathways and differentially expressed genes associated with lymphocystis nodule development caused by LCDV infection in flounder for the first time, providing a new breakthrough in molecular mechanisms of lymphocystis formation in fish.

Seasonal variations in the diversity and benthic community structure of subtidal artificial oyster reefs adjacent to the Luanhe River Estuary, Bohai Sea.

Artificial oyster reefs provide important spawning and nursery grounds for a variety of fishes and large mobile crustaceans. Between July 2016 and May 2017, seasonal surveys of species composition and community structure were performed in the artificial oyster reef area and control area adjacent to the Luanhe River Estuary in China. During the survey year, 56 species belonging to 50 genera, 45 families, and 19 orders were recorded. The dominant economically important fish and mobile crustaceans were Hexagrammos otakii, Pholis fangi, Sebastes schlegelii, Charybdis japonica, and Oratosquilla oratoria. Resident fishes belonged to the Cynoglossidae, Paralichthyidae, Pleuronectidae, and Gobiidae families. Seasonally important fish species included Lateolabrax japonicus, Konosirus punctatus, Thryssa kammalensis, Hexagrammos agrammus, and Acanthopagrus schlegelii. The ranges of H' values among stations were 1.18-2.16, 0.65-1.75, 1.18-2.06, and 0.62-1.92 in spring, summer, autumn, and winter, respectively. The benthic organisms present in the community of artificial oyster reef areas can be classified into groups according to month and season. The abundance biomass curves showed that the oyster reef area in spring, autumn, and winter experienced low disturbance, whereas the community structure in summer was subject to large variations from external disturbance. We also found that as the age of the oyster reefs increased, the percentage of oysters in the low shell height group (< 40 mm) decreased. The oyster density was 324 ind/m2 for the reef created in 2016, 724 ind/m2 for the reef created in 2015, and 364 ind/m2 for the reef created in 2013. These findings can be used to develop suitable management strategies for the sustainable maintenance of artificial oyster reef ecosystems.

Anti-microbial activity and immunomodulation of recombinant hepcidin 2 and NK-lysin from flounder (Paralichthys olivaceus).

Infections due to pathogens impact global aquaculture economy, where diseases caused by bacteria should be in particular focus due to antibiotic resistance. Hepcidin and NK-lysin are important innate immune factors having potential to be exploited as alternatives to antibiotics due to their antimicrobial activity and immunomodulatory capacity. In this study, the recombinant proteins of hepcidin 2 and NK-lysin (rPoHep2 and rPoNKL) from flounder (Paralichthys olivaceus) were obtained via a prokaryotic expression system. The results exhibited that rPoHep2 and rPoNKL killed both gram-negative and gram-positive bacteria mainly via attachment and disruption of the membrane. Interestingly, both peptides could bind to bacterial DNA. The antiviral assay showed that both peptides have antiviral activity against hirame nonvirhabdovirus. They exhibited no cytotoxicity to the mammalian and fish cell lines. PoHep2 was found localized in G-CSFR-positive peritoneal cells. Moreover, rPoHep2 significantly enhanced the phagocytosis of flounder leukocytes in vitro. These findings suggested that neutrophils contained rPoHep2 and may respond to the immunoreaction of neutrophils. In summary, both rPoHep2 and rPoNKL possess antimicrobial activities and may be exploited to replace traditional antibiotics. rPoHep2 possess immune regulatory functions, that can be further investigated as an immunostimulant in aquaculture.

An antiviral optimized extract from Sanguisorba officinalis L. roots using response surface methodology, and its efficacy in controlling viral hemorrhagic septicemia of olive flounder (Paralichthys olivaceus).

Viral hemorrhagic septicemia causes considerable economic losses for Korea's olive flounder (Paralichthys olivaceus) aquaculture farms; therefore, effective antiviral agents for controlling viral hemorrhagic septicemia virus (VHSV) infection are imperative. The present study implemented a Box-Behnken design and cytopathic reduction assay to derive an optimized extract of Sanguisorba officinalis L. roots (OE-SOR) with maximum antiviral activity against VHSV. OE-SOR prepared under optimized extraction conditions (55% ethanol concentration at 50 °C for 5 h) exhibited potent antiviral activity against VHSV, with a 50% effective 0.21 µg/mL concentration and a 340 selective index. OE-SOR also showed direct virucidal activity in the plaque reduction assay. Administering OE-SOR to olive flounder exhibited substantial efficacies against VHSV infection. Fish receiving 100 mg/kg body weight/day of OE-SOR as a preventive (40.0%; p < 0.05) or therapeutic (44.4%; p < 0.05) exhibited a higher relative survival than the untreated VHSV-infected control group (mortalities of 100% and 90%, respectively). In addition, fish fed with OE-SOR (100 mg/kg body weight/day) for two weeks conveyed a significantly higher inflammatory cytokine expression (nuclear factor kappa-light-chain-enhancer of activated B cells [NF-κB], interleukin-1 beta [IL-1ß], and tumor necrosis factor-alpha [TNF-α]) than the control group one to two days post-administration. Moreover, no hematological or histological changes were observed in olive flounder treated with OE-SOR over four weeks. Liquid chromatography-quadrupole-time of flight tandem mass spectrometry and -triple quadrupole tandem mass spectrometry analyses identified ziyuglycoside I as a prominent OE-SOR constituent and marker compound (content: 14.5%). This study verifies that OE-SOR is an effective alternative for controlling viral hemorrhagic septicemia in olive flounder farms as it exhibits efficient in vivo anti-VHSV activity and increases innate immune responses.

Molecular cytogenetic analysis of the olive flounder embryonic cell line FGBC8 and its applicability to biotechnology.

We explored the biotechnological applicability of a previously established olive flounder (Paralichthys olivaceus) embryonic cell line (FGBC8). FGBC8 was transfected with pEGFP-c1 and pluripotency-related genes, then infected with viral hemorrhagic septicemia virus (VHSV), and the expression of immune-related genes was observed through quantitative real-time polymerase chain reaction. Transfected cells showed strong green fluorescence 48 h after transfection, and pluripotency-related genes were successfully transfected. In addition, FGBC8 cells were highly susceptible to VHSV and the expression of immune-related genes was induced during infection. Our results demonstrate that FGBC8 cells are valuable research tools for assessing host-pathogen interactions and biotechnological applications.

Establishment and Characterization of Continuous Satellite Muscle Cells from Olive Flounder (Paralichthys olivaceus): Isolation, Culture Conditions, and Myogenic Protein Expression.

Olive flounder (Paralichthys olivaceus) muscle satellite cells (OFMCs) were obtained by enzymatic primary cell isolation and the explant method. Enzymatic isolation yielded cells that reached 80% confluence within 8 days, compared to 15 days for the explant method. Optimal OFMC growth was observed in 20% fetal bovine serum at 28 °C with 0.8 mM CaCl2 and the basic fibroblast growth factor (BFGF) to enhance cell growth. OFMCs have become permanent cell lines through the spontaneous immortalization crisis at the 20th passage. Olive flounder skeletal muscle myoblasts were induced into a mitogen-poor medium containing 2% horse serum for differentiation; they fused to form multinucleate myotubes. The results indicated complete differentiation of myoblasts into myotubes; we also detected the expression of the myogenic regulatory factors myoD, myogenin, and desmin. Upregulation (Myogenin, desmin) and downregulation (MyoD) of muscle regulation factors confirmed the differentiation in OFMCs.

Mechanisms underlying probiotic effects on neurotransmission and stress resilience in fish via transcriptomic profiling.

In recent years, studies have highlighted the significant impact of probiotic treatment on the central nervous system (brain) and stress regulation through the microbiota-gut-brain axis, yet there have been limited knowledge on this axis in fish. Therefore, this study aimed to enhance the current understanding of the mechanisms underlying probiotic effects on neurotransmission and stress alleviation in fish through transcriptomic profiling. In this study, olive flounders (Paralichthys olivaceus) were subjected to two trial setups: a 1-month lab-scale trial and a 6-month field-scale trial, with and without the probiotic strain Lactococcus lactis WFLU12. RNA-Seq analysis was performed using liver samples collected from fish at one-month post-feeding (mpf) in both trials. Additionally, fish growth was monitored monthly, and serological parameters were measured at one mpf in the field-scale experiment. The results of the lab-scale trial showed that probiotic administration significantly upregulated genes related to neurotransmission, such as htr3a, mao, ddc, ntsr1, and gfra2. These findings highlight the impact of probiotics on modulating neurotransmission via the microbiota-gut-brain axis. In the field-scale experiment, fish growth was significantly promoted and the sera levels of AST, LDH, and cortisol were significantly higher in the control group compared to the probiotics group. Furthermore, genes involved in stress responses (e.g. hsp70, hsp90B1, hspE1, prdx1, and gss) and transcriptional regulators (e.g. fos, dusp1, and dusp2) exhibited significant upregulation in the control group compared to the probiotics group, indicating that probiotic administration can alleviate stress levels in fish. Overall, this study provides valuable insights into the mechanisms underlying the beneficial effects of probiotics in fish, specifically regarding their impact on neurotransmission and stress alleviation.

Characterisation, evolution and expression analysis of the interferon regulatory factor (IRF) family from olive flounder (Paralichthys olivaceus) in response to Edwardsiella tarda infection and temperature stress.

Interferon regulatory factor (IRF) family involves in the transcriptional regulation of type I Interferons (IFNs) and IFN-stimulated genes (ISGs) and plays a critical role in cytokine signaling and immune response. However, systematic identification of the IRF gene family in teleost has been rarely reported. In this study, twelve IRF members, named PoIRF1, PoIRF2, PoIRF3, PoIRF4a, PoIRF4b, PoIRF5, PoIRF6, PoIRF7, PoIRF8, PoIRF9, PoIRF10 and PoIRF11, were identified from genome-wide data of olive flounder (Paralichthys olivaceus). Phylogenetic analysis indicated that PoIRFs could be classified into four clades, including IRF1 subfamily (PoIRF1, PoIRF11), IRF3 subfamily (PoIRF3, PoIRF7), IRF4 subfamily (PoIRF4a, PoIRF8, PoIRF9, PoIRF10) and IRF5 subfamily (PoIRF5, PoIRF6). They were evolutionarily related to their counterparts in other fish. Gene structure and motif analysis showed that PoIRFs protein sequences were highly conserved. Under normal physiological conditions, all PoIRFs were generally expressed in multiple developmental stages and healthy tissues. After E. tarda attack and temperature stress, twelve PoIRFs showed significant and different changes in mRNA levels. The expression of PoIRF1, PoIRF3, PoIRF4a, PoIRF5, PoIRF7, PoIRF8, PoIRF9, PoIRF10 and PoIRF11 could be markedly induced by E. tarda, indicating that they played a key role in the process of antibacterial immunity. Besides, temperature stress could significantly stimulate the expression of PoIRF3, PoIRF5, PoIRF6 and PoIRF7, indicating that they could transmit signals rapidly when the temperature changes. In conclusion, this study reported the molecular properties and expression analysis of PoIRFs, and explored their role in immune response, which laid a favorable foundation for further studies on the evolution and functional characteristics of the IRF family in teleost fish.

Establishment and identification of a skin cell line from Chinese tongue sole (Cynoglossus semilaevis) and analysis of the changes in its transcriptome upon LPS stimulation.

The Chinese tongue sole (Cynoglossus semilaevis) holds significant economic importance within the fishing industry along the eastern coasts of China. In recent years, the frequent outbreaks of bacterial diseases have become a common concern as the aquaculture scale expands. The majority of the diseased fish exhibit symptoms such as skin congestion, damage and skin ulceration. As the skin serves as the first line of defense against bacterial infections, establishing a skin cell line for immunological research on Chinese tongue sole's response to bacterial infection is of utmost importance. In this study, a cell line named CSS (derived from the skin of the Chinese tongue sole) was successfully established. The cells have demonstrated stability during passages and exhibit a multipolar fibroblast-like morphology. They were cultured in L-15 medium with 20% serum and have been successfully passed through 60 passages over a period of 20 months. The identification of the mitochondrial CO1 gene confirmed that the cell originated from Chinese tongue sole. The karyotype detection revealed that the cell had a chromosome number of 2n = 42. After being stored in liquid nitrogen for 15 months, the cells can maintain more than 75% viability upon recovery. After transfecting with cy3-labeled scramble siRNA and pEGFP-N3 plasmid, clear fluorescence was observed in the transfected cells. We observed that lipopolysaccharide (LPS) from Escherichia coli significantly upregulate the gene expression of various immune-related pathways at 2 h in the CSS cell line. Additionally, the differentially expressed genes showed a higher enrichment in immune-related pathways at 2 and 6 h after stimulation compared to the 24 h point. Moreover, we identified 347 genes that exhibited a gradual increase in expression during the 0-24 h stimulation period. These genes were primarily enriched in pathways related to Autophagy, GABAergic synapse, Apelin signaling and Ferroptosis. In general, the CSS cell line established in this study exhibits stable growth and can serve as a valuable tool for in vitro studies of immunology and other basic biologies of Chinese tongue sole.

The regulatory function of GATA3 on immune response in Japanese flounder (Paralichthys olivaceus).

GATA3 belongs to the GATA family, and it could interact with the target gene promoter. It has been reported to play a central role in regulating lymphocyte differentiation. In this study, the GATA3 cDNA sequence was identified by a homologous clone and the RACE technology from Japanese flounder (Paralichthys olivaceus). The full-length of the GATA3 cDNA sequence was 2904 bp, including 1332 bp open reading frame (ORF), 265 bp 5 '-untranslated region (5' UTR), and 1308 bp 3 '-UTR, encoding 443 amino acids. GATA3 protein sequence was conserved in vertebrates and invertebrates, including two zinc finger domains. qRT-PCR showed that the expression of GATA3 was high in the gill, kidney, and spleen. Expression of GATA3 slowly increased at the earlier stages and culminated at the late gastrula and somatic stages. Immunohistochemistry (IHC) results showed that the GATA3 protein was expressed in lymphocyte cells, undifferentiated basal and pillar cells of the gills, as well as lymphocyte cells and melanin macrophages of the kidney. The expression of GATA3 was significantly regulated in tissues and different types of lymphocytes after stimulation with Edwardsiella tarda. Dual-luciferase reporter assay indicated that the GATA3 protein could directly interact with promoters of target genes involved in the immune response. These findings suggested that GATA3 plays a major role in regulating the immune response. This study provided a theoretical basis for the immune response mechanism of teleost and a useful reference for later research on fish immunology.

Untargeted LC-MS metabolomics reveals the metabolic responses in olive flounder subjected to hirame rhabdovirus infection.

Hirame novirhabdovirus (HIRRV), which mainly infects the olive flounder (Paralichthys olivaceus), is considered to be one of the most serious viral pathogens threatening the global fish culture industry. However, little is known about the mechanism of host-pathogen interactions at the metabolomic level. In this study, in order to explore the metabolic response of olive flounder to HIRRV infection, liquid chromatography mass spectrometry (LC-MS) was used to detect the changes of endogenous compounds of the olive flounder after HIRRV infection. A total of 954 unique masses were obtained, including 495 metabolites and 459 lipids. Among them, 7 and 173 qualified differential metabolites were identified at 2 days and 7 days post-infection, respectively. Distinct metabolic profiles were observed along with viral infection. At the early stage of infection, only a few metabolites were perturbed. Among them, the level of inosine and carnosine were increased and the potential antiviral ability of these two metabolites was further confirmed by exogenous addition experiment. At the late stage of HIRRV infection, the metabolic profiles changed remarkably. The changes in amino acids and nucleotides especially the 7-methylguanine also accelerated the amplification of viral particles. And the down-regulation of glutathione (GSH) implied an elevated level of ROS (reactive oxygen species) that attenuated the immune system of flounders. HIRRV also induced the accumulation of purine and reduction of pyrimidine, and elevated LPC and LPE levels. The unbalanced purine/pyrimidine and altered lipid profile may be beneficial for the replication and infection of HIRRV at the late stage of infection. These findings provide new insights into the pathogenic mechanism of HIRRV infection in olive flounder.

Hidden in the fog: morphological and molecular characterisation of Derogenes varicus sensu stricto (Trematoda, Derogenidae) from Sweden and Norway, and redescription of two poorly known Derogenes species.

Derogenes varicus (Müller, 1784) is widely reported as a trematode with exceptionally low host specificity and a wide, bipolar distribution. However, several recent studies have suggested that D. varicus represents a species complex and based on molecular evidence, four genetic lineages (labeled as "DV1-4") have been designated within the D. varicus species complex. This possibility requires improved (ideally molecular) characterisation of specimens from the type-host (Salmo salar) and type-locality (off Denmark). During examination of trematode parasites of fish from Scandinavian and Arctic waters (Sweden and Norway), we found specimens of D. varicus in the stomach of Merlangius merlangus off the coast of Sweden, and in Gadus morhua off the coast of Sweden and Norway; we compared them to D. varicus from the type-host, the Atlantic salmon Salmo salar from Norway, to verify their conspecificity. Newly generated sequences (28S rDNA, ITS2 and cox1) of Scandinavian and Arctic specimens consistent with D. varicus all formed a single clade, DV1. 28S sequences of D. varicus from S. salar from Norway, i.e., close to the Danish type locality, clustered within the DV1 clade along with sequences of D. varicus from various hosts including Limanda limanda, G. morhua and Myoxocephalus scorpius from the White Sea and the Barents Sea (Russia), without any host-related structuring. We thus consider that the lineage DV1 represents D. varicus sensu stricto. Additionally, specimens from M. merlangus had a similar morphology and anatomy to those of D. varicus from L. limanda, G. morhua and M. scorpius from T. Odhner's collection, supporting the presence of a single species in the DV1 lineage designated herein as D. varicus sensu stricto. We redescribe D. varicus sensu stricto, add new morphological characters and provide morphometric data. We infer that D. varicus types DV2-4 all relate to separate species. We also revise type-specimens of Derogenes minor Looss, 1901 from the A. Looss collection in the Swedish Museum of Natural History and provide redescriptions of it and of the type-species of the genus, Derogenes ruber Lühe, 1900. In light of their morphological distinctiveness relative to D. varicus sensu stricto, we reinstate D. parvus Szidat, 1950 and D. fuhrmanni Mola, 1912.

Title: Caché dans le brouillard : caractérisation morphologique et moléculaire de Derogenes varicus sensu stricto (Trematoda, Derogenidae) de Suède et de Norvège, et redescription de deux espèces de Derogenes peu connues. Abstract: Derogenes varicus (Müller, 1784) est largement signalé comme un trématode avec une spécificité d'hôte exceptionnellement faible et une large distribution bipolaire. Cependant, plusieurs études récentes ont suggéré que D. varicus représente un complexe d'espèces. Cette possibilité nécessite une meilleure caractérisation (idéalement moléculaire) des spécimens de l'hôte-type (Salmo salar) et de la localité-type (au large du Danemark). Lors de l'examen des trématodes parasites des poissons des eaux scandinaves et arctiques (Suède et Norvège), nous avons trouvé des spécimens de D. varicus dans l'estomac de Merlangius merlangus au large de la Suède, et dans Gadus morhua au large de la Suède et de la Norvège ; nous les avons comparés à D. varicus de l'hôte-type, le saumon atlantique Salmo salar de Norvège, pour vérifier leur conspécificité. Les séquences nouvellement générées (ADNr 28S, ITS2 et cox1) de spécimens scandinaves compatibles avec D. varicus ont toutes formé un seul clade, DV1. Les séquences 28S de D. varicus de S. salar de Norvège, c'est-à-dire proches de la localité-type au Danemark, se regroupent dans le clade DV1 avec des séquences de D. varicus de divers hôtes dont Limanda limanda, G. morhua et Myoxocephalus scorpius de la mer Blanche et la mer de Barents (Russie), sans aucune structuration liée à l'hôte. Nous considérons donc que la lignée DV1 représente D. varicus sensu stricto. De plus, les spécimens de M. merlangus avaient une morphologie et une anatomie similaires à celles de D. varicus de L. limanda, G. morhua et M. scorpius de la collection de T. Odhner, confirmant la présence d'une seule espèce dans la lignée DV1 désignée ici comme D. varicus sensu stricto. Nous redécrivons D. varicus sensu stricto, ajoutons de nouveaux caractères morphologiques et fournissons des données morphométriques. Nous en déduisons que les types DV2-4 de D. varicus se rapportent tous à des espèces distinctes. Nous révisons également les spécimens-types de Derogenes minor Looss, 1901 de la collection d'A. Looss du Muséum Suédois d'Histoire Naturelle, et fournissons des redescriptions de cette espèce et de l'espèce-type du genre, Derogenes ruber Lühe, 1900. À la lumière de leur distinction morphologique par rapport à D. varicus sensu stricto, nous rétablissons D. parvus Szidat, 1950 et Derogenes fuhrmanni Mola, 1912.