Effect of creatine administration on locomotor activity and stress response in olive flounder (Paralichthys olivaceus).

The creatine kinase system is crucial for maintaining cellular energy homeostasis and plays a role in regulating locomotor behavior in organisms, but its significance in the regulating the motionless behavior in olive flounder is limited. In the first experiment of this study, elevated levels of creatine kinase (CK) activity in the spinal cord were detected in the juvenile group (JG) flounder compared to the adult group (AG) flounder. In the second experiment, to further confirm the involvement of CK in the locomotor behavior, the adult flounder was given an intraperitoneal injection of creatine (150 mg/kg), while the flounder in the control group received a saline solution. After one week post-injection, the behavioral analysis revealed that the flounder in the creatine-treated group displayed higher levels of locomotor activity and a greater number of escape attempts in response to external stimuli when compared to the control group. However, the acute stress response, induced by intraperitoneal injection and characterized by tail beating, was significantly alleviated in the flounder in the creatine-treated group. Additionally, there was an upregulation of the UII and AchR genes in the spinal cord, as well as increased levels of UII and AchR in the muscle tissues of the creatine-treated flounder. However, a reduction in UI mRNA levels was observed in the brain of the flounder. Collectively, our data provide the evidence that the elevated enzyme activity and gene expression of creatine kinase play important roles in off-bottom swimming behavior in the JG flounder. Furthermore, administration of creatine improved the locomotor activity and alleviated the stress response in flounder, which is associated with regulation of the locomotor- and stress-related gene in the brain, spinal cord, and muscle.

Antiviral effects of umbelliferone against viral hemorrhagic septicemia virus in olive flounder (Paralichthys olivaceus).

Viral hemorrhagic septicemia virus (VHSV) poses a significant threat to global aquaculture, prompting ongoing efforts to identify potential drug candidates for disease prevention. Coumarin derivatives have recently emerged as a promising class of compounds effective against rhabdoviruses, which severely impact the aquaculture industry. In this study, we assessed the anti-VHSV activity of umbelliferone (7-hydroxycoumarin) in fathead minnow (FHM) cells and olive flounder Paralichthys olivaceus. Umbelliferone exhibited an EC50 of 100 µg/mL by reducing cytopathic effect, with a maximum cytotoxicity of 30.9 % at 750 µg/mL. Mechanistic analyses via a time-course plaque reduction assay revealed that direct incubation with the virus for 1 h resulted in 97.0 ± 1.8 % plaque reduction, showing excellent direct virucidal activity. Pretreatment for 4 h resulted in a 33.5 ± 7.8 % plaque reduction, which increased with longer incubation times. Cotreatment led to a 33.5 ± 2.9 % plaque reduction, suggesting interference with viral binding, whereas postinfection treatment proved less effective. Umbelliferone was prophylactically administered to the olive flounder through short-term (3 days) and long-term (14 days) medicated feeding, followed by a 4-day postinfection period. Short-term administration at 100 mg/kg body weight (bw)/day resulted in the highest relative percent survival (RPS) of 56 %, whereas long-term administration achieved a maximum RPS of 44 % at 30 mg/kg bw/day. Umbelliferone administration delayed mortality at these doses. Additionally, umbelliferone significantly inhibited the expression of the VHSV N gene during viral challenge, with no observed toxic effects in fish up to an administration dose of 30 mg/kg bw/day for 28 days. Our findings suggest that the protective mechanism of short-term administration of 100 mg umbelliferone against VHSV infection may involve the overexpression of TLR2, MDA5, STAT1, and NF-κB at 24 h postinfection (hpi). IL-8 and IFN II expression was upregulated, whereas TNF-α, IL-1ß, and IFN I expression was suppressed at 24 hpi. The upregulation of ISG15 at 48 hpi may contribute to the inhibition of VHSV replication, whereas the downregulation of Caspase 3 expression at 96 hpi suggests a possible inhibition of virus-induced apoptosis at later infection stages. Overall, umbelliferone exhibited anti-VHSV activity through multiple mechanisms, with the added advantage of convenient administration via medicated feed.

Evaluation of Gamma-Aminobutyric Acid (GABA) as a Functional Feed Ingredient on Growth Performance, Immune Enhancement, and Disease Resistance in Olive Flounder (Paralichthys olivaceus) under High Stocking Density.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid that is found in the brain and central nervous system of animals as an inhibitory neurotransmitter. It has been shown to have a variety of physiological functions, including stress reduction and immune enhancement. This study investigated the effects of dietary supplementation with GABA on growth, serum biochemistry, innate immunity, and disease resistance in juvenile olive flounders (Paralichthys olivaceus) challenged with Edwardsiella tarda under high-stocking density. A control diet and three experimental diets were prepared, with 150 mg/kg (GABA150), 200 mg/kg (GABA200), and 250 mg/kg (GABA250) of GABA added to each diet, respectively. Each experimental diet was fed to olive flounders in triplicate with an initial weight of 12.75 g ± 0.3 g in 40 L tanks at two stocking densities: normal density (20 fish/tank) and high density (40 fish/tank). After 8 weeks of the feeding trial, growth, feed utilization, whole-body proximate compositions, blood analyses, and non-specific immune responses were measured, and challenge tests were performed. There were no significant differences in the weight gain (WG) and specific growth rate (SGR) among fish fed the GABA-supplemented diets at the two stocking densities. However, the normal-density groups showed significantly higher WG and SGR than the high-density groups (p < 0.05). There was no significant difference in feed efficiency and protein efficiency ratio among all groups. Moreover, there was no significant difference in the whole-body proximate composition analysis (p > 0.05). There were no significant differences in cortisol levels in fish fed the GABA at both densities, but the high-density group showed a significantly higher cortisol than the low-density group. Blood GABA significantly increased in a dose-dependent manner regardless of the density groups (p < 0.05). Superoxide dismutase activity showed significantly higher levels than the control group, but there was no significant effect of the stocking densities in fish fed the GABA diets (p < 0.05). Myeloperoxidase activities in fish fed the GABA200 and GABA250 diets showed significantly higher levels at both of the stocking densities (p < 0.05). Lysozyme activity was significantly higher in the GABA150 group than in the CON, GABA200, and GABA250 groups (p < 0.05). After 15 days of challenge tests with Edwardsiella tarda, the cumulative survival rates of the GABA150, GABA200, and GABA250 groups were significantly higher than that of the CON group (p < 0.05). The results suggested that the optimal dietary GABA level for juvenile olive flounder culture is 150 mg/kg, regardless of rearing density, to enhance growth, immunity, and disease resistance.

Olive Flounder By-Product Prozyme2000P Hydrolysate Ameliorates Age-Related Kidney Decline by Inhibiting Ferroptosis.

This study explores olive flounder by-product Prozyme2000P (OFBP) hydrolysate as a potential treatment for age-related kidney decline. Ferroptosis, a form of cell death linked to iron overload and oxidative stress, is increasingly implicated in aging kidneys. We investigated whether OFBP could inhibit ferroptosis and improve kidney health. Using TCMK-1 cells, we found that OFBP treatment protected cells from ferroptosis induced by sodium iodate (SI). OFBP also preserved the mitochondria health and influenced molecules involved in ferroptosis regulation. In aging mice, oral administration of OFBP significantly improved kidney health markers. Microscopic examination revealed reduced thickening and scarring in the kidney's filtering units, a hallmark of aging. These findings suggest that OFBP hydrolysate may be a promising therapeutic candidate for age-related kidney decline. By inhibiting ferroptosis, OFBP treatment appears to improve both cellular and structural markers of kidney health. Further research is needed to understand how OFBP works fully and test its effectiveness in more complex models.

Association Between Muscle Growth and Transcription of a Mutant MSTN Gene in Olive Flounder (Paralichthys olivaceus).

Myostatin (MSTN, also known as growth differentiation factor-8 (GDF-8)), a member of the transforming growth factor ß (TGF-ß) superfamily, functions as a negative regulator of skeletal muscle development and growth. However, it is also expressed in a wide range of tissues in fish and thus may have more diverse roles in this group than in mammals. In this study, we assessed the genome-wide transcriptional expression pattern associated with the CRISPR/Cas9-mutated MSTN gene in the olive flounder (Paralichthys olivaceus) in association with changes in cell proliferation and transportation processes. There were no differences in the hepatosomatic index, and the growth of male and female fish increased in the F1 progeny of the MSTN mutants. Furthermore, the histopathological analysis showed that myostatin editing resulted in a 41.24% increase in back muscle growth and 46.92% increase in belly muscle growth in male flounder compared with normal flounder, and a 16.01% increase in back muscle growth and 14.26% increase in belly muscle growth in female flounder compared with normal flounder. This study demonstrates that editing of the myostatin gene enhances muscle growth in olive flounder, with a notably more pronounced effect observed in males. Consequently, myostatin-edited male flounder could represent a valuable asset for the flounder aquaculture industry.

Exosomes derived from olive flounders infected with Streptococcus parauberis: Proteomic analysis, immunomodulation, and disease resistance capacity.

Multidrug-resistant Streptococcus parauberis causes high fish mortality in aquaculture, necessitating an urgent need for innovative control strategies. This study aimed to develop an immunizing agent against S. parauberis using exosomes isolated from the plasma of olive flounders infected experimentally with S. parauberis (Sp-Exo). Initially, we tested the in vitro immunomodulatory effect of Sp-Exo in murine macrophage RAW264.7 cells and compared it to that of exosomes isolated from naïve fish (PBS-Exo-treated). Notably, Sp-Exo treatment significantly (p < 0.05) upregulated pro-and anti-inflammatory cytokines (Il1ß, Tnfα, and Il10), antimicrobial peptide, defensin isoforms (Def-rs2 and Def-ps1), and antiviral (Ifnß1 and Isg15) genes. In vivo studies in larval and adult zebrafish revealed similar patterns of immunomodulation. Furthermore, larval and adult zebrafish exhibited significantly (p < 0.05) enhanced resistance to S. parauberis infection following treatment with Sp-Exo compared to that with PBS-Exo. Proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) approach revealed the presence of 77 upregulated and 94 downregulated differentially expressed proteins (DEPs) in Sp-Exo, with 22 and 37 significantly (p < 0.05) upregulated and downregulated DEPs, respectively. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Search Tool for the Retrieval of Interacting Genes/Proteins analyses revealed that these genes are associated with key pathways, such as innate immune responses, complement system, acute phase responses, phospholipid efflux, and chylomicron remodeling. In conclusion, Sp-Exo demonstrated superior immunomodulatory activity and significant resistance against S. parauberis infection relative to that on treatment with PBS-Exo. Proteomic analysis further verified that most DEPs in Sp-Exo were associated with immune induction or modulation. These findings highlight the potential of Sp-Exo as a promising vaccine candidate against S. parauberis and other bacterial infections in olive flounder.

α1 and ß3 adrenergic receptor-mediated excitatory effects of adrenaline on the caudal neurosecretory system (CNSS) in olive flounder, Paralichthys olivaceus.

Adrenaline is one of the most important neurotransmitters in the central nervous system and is produced during stress. In this study, we investigated the modulatory role of adrenaline and adrenergic receptors on the neuroendocrine Dahlgren cells in the caudal neurosecretory system (CNSS) of olive flounder. Ex vivo electrophysiological recordings revealed that adrenaline significantly increased the firing frequency and altered the firing pattern of Dahlgren cells. Moreover, treatment with adrenaline led to a significant upregulation of ion channels and major hormone secretion genes in CNSS at the mRNA levels. Additionally, treatment with adrenaline resulted in a significantly elevation in the expression levels of α1- and ß3-adrenergic receptors. Furthermore, the ß3-adrenergic receptor antagonist exerts a significant inhibitory effect on adrenaline-induced enhancement firing activities of Dahlgren cells, whereas the α1-adrenergic receptor antagonist displays a comparatively weaker inhibitory effect. Additionally, the enhanced firing activity induced by adrenaline could be effectively suppressed by both α1- and ß3-adrenergic receptor antagonists. Taken together, these findings provide strong evidence in favor of the excitatory effects of adrenaline through α1 and ß3 adrenergic receptors in CNSS to stimulate the secretion of stress-related hormones, ß3-adrenergic receptor plays a more dominant role in the modulation of firing activities of Dahlgren cells by adrenaline and thereby regulates the stress response in olive flounder.

The residue of salinomycin in the muscles of olive flounder (Paralichthys olivaceus) and black rockfish (Sebastes Schlegeli) after oral administration analyzed by LC-Tandem-MS.

BACKGROUND: Salinomycin, an antibiotic, have potential as a veterinary drug for fish due to its anti-parasitic activity against several fish parasites. Thus the residual levels of salinomycin in muscles of two significant aquaculture species in Korea, olive flounder and black rockfish, were analyzed using HPLC-MS-MS. RESULTS: The proper method to analyze the residual salinomycin in fish muscles using LC-MS-MS was settled and the method was validated according to CODEX guidelines. The residues in three distinct groups for two fish species were analyzed using the matrix match calibration curves at points of five different times following oral administration. After oral administration, salinomycin rapidly breaks down in both olive flounder and black rockfish. After 7th days, the average residue in all groups of two fish spp. decreased below limit of quantitation (LOQ). CONCLUSION: Due to low residue levels in fish muscles, salinomycin may therefore be a treatment that is safe for both fish and humans. This result could contribute to establishment of MRL (minimal residual limit) for approval of salinomycin for use in aquaculture.

Draft genome sequence of multiple antibiotic-resistant Vibrio harveyi isolated from diseased olive flounder (Paralichthys olivaceus) farmed in South Korea.

Vibrio harveyi strain 22FBVib0145 was isolated from a diseased olive flounder farmed in Jeju, Korea. Here, we report the draft genome sequence of this strain. It is 6,238,277 bp in length with a G + C content of 44.8%.

Whole genome identification of olive flounder (Paralichthys olivaceus) cathepsin genes: Provides insights into its regulation on biotic and abiotic stresses response.

Cathepsins are major lysosomal enzymes involved in essential physiological processes, including protein degradation, tissue differentiation, and innate or adaptive responses. Several kinds of cathepsins have been reported in teleost fishes, but no characterization have been performed for the inflammatory response of cathepsin family in olive flounder until now. In our current study, a total of 17 cathepsins in olive flounder were systematically identified and characterized. Phylogenetic analysis clearly indicated that the cathepsin genes was highly conserved. Analysis of structure and motifs exhibited high sequence similarity of cathepsin genes in olive flounder. Expression profiles of cathepsin genes in different tissues and developmental stages showed that cathepsins were temporally and spatially specific. RNA-seq analysis of bacteria and temperature stresses revealed that members of cathepsin were involved in inflammatory responses. Collectively, our findings would provide a further reference for understanding the molecular mechanisms of cathepsins in olive flounder.

Determination of the minimum infective dose of viral hemorrhagic septicemia virus (VHSV) in juvenile olive flounder, Paralichthys olivaceus using an immersion challenge model.

Viral hemorrhagic septicemia virus (VHSV) affects over 80 fish species, leading to viral hemorrhagic septicemia (VHS). Horizontal VHSV transmission is widely studied, with researchers utilizing various doses to establish infection models. Infected hosts shed the virus into the environment, elevating the risk of transmission to naïve fish within the same system. This study aimed to ascertain the minimum infective dose of VHSV in olive flounder (Paralichthys olivaceus). In olive flounder, the detection of VHSV within the kidney exhibited the highest infection rate on the third day among days 1, 3 and 5. Doses of 103.0 to 104.7 TCID50/ml were administered to juvenile olive flounder across three farms. Results showed resistance to infection below 103.4 TCID50/ml at 15 °C. While infection frequency varied by concentration, higher concentrations correlated with more infections. Nonetheless, viral copy numbers did not differ significantly among infected fish at varying concentrations. This study underscores the need for early VHSV management and contributes essential data for pathogenicity assessment and foundational knowledge.

Combination of Host-Associated Rummeliibacillus sp. and Microbacterium sp. Positively Modulated the Growth, Feed Utilization, and Intestinal Microbial Population of Olive Flounder (Paralichthys olivaceus).

Two novel strains of Rummeliibacillus sp. and Microbacterium sp. were identified from the intestine of olive flounder (Paralichthys olivaceus) and characterized in vitro as potential probiotics. Feeds without probiotic and with a 50:50 mixture of these two strains (1 × 108 CFU/g feed) were denoted as the control and Pro diets, respectively. Three randomly selected tanks (20 flounders/tank, ~11.4 g each) were used for each diet replication. After 8 weeks of feeding, the growth and feed utilization of the flounder in the Pro group improved (p < 0.05) compared to the control. Among four immune parameters, only myeloperoxidase activity was elevated in the Pro group. Serum biochemistry, intestinal microbial richness (Chao1), and diversity (Shannon index) remained unchanged (p ≥ 0.05), but phylogenetic diversity was enriched in the Pro fish intestine. Significantly lower Firmicutes and higher Proteobacteria were found in the Pro diet; the genus abundance in the control and Pro was as follows: Staphylococcus > Lactobacillus > Corynebacterium and Lactobacillus > Staphylococcus > Corynebacterium, respectively. Microbial linear discriminant scores and a cladogram analysis showed significant modulation. Therefore, the combination of two host-associated probiotics improved the growth and intestinal microbial population of flounder and could be supplemented in the Korean flounder industry.

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.

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.

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.

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 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.

Effect of Four Functional Feed Additives on Growth, Serum Biochemistry, Antioxidant Capacity, Gene Expressions, Histomorphology, Digestive Enzyme Activities and Disease Resistance in Juvenile Olive Flounder, Paralichthys olivaceus.

An 8-week feeding trial was executed to evaluate the efficacy of four functional feed additives in replacing antibiotics in juvenile olive flounder, Paralichthys olivaceus, fed with a low-fish-meal diet. A basal diet without feed additives was used as a control (CON); other diets were formulated by supplementing 0.50% taurine (TW), 0.30% peptide (PT), 0.23% mineral water (MW), 0.35% yeast-extracted nucleotides (GRO), 0.35% GRO + 0.50% taurine (GROTW), 0.35% GRO + 0.30% peptide (GROPT) and 0.35% GRO + 0.23% mineral water (GROMW) into the basal diet; in addition, one diet was supplemented with oxytetracycline (OTC) at 0.5% as a positive control. Triplicate groups of 25 fish with an average weight of 5.15 ± 0.06 g (mean ± SD) were fed one of the nine experimental diets. At the end of the feeding trial, the weight gain, specific growth rate and protein efficiency ratio of fish fed the GRO, GROMW, GROPT and GROTW diets were significantly higher than those of fish fed the CON diet (p < 0.05). The feed efficiency of fish fed the GRO, GROMW, GROPT and GROTW diets was significantly higher than that of fish fed the TW and OTC diets. However, the survival, hepatosomatic index, viscerosomatic index and condition factor of fish, as well as their whole-body proximate composition, were not significantly affected by the experimental diets (p > 0.05). The serum glutamic pyruvic transaminase of fish fed the GROPT diet was significantly lower than that of fish fed the CON diet. However, glutamic oxaloacetic transaminase, glucose and total protein were not significantly affected by the experimental diets (p > 0.05). The serum superoxide dismutase activity of fish fed the PT, TW, GRO, GROMW, GROPT and GROTW diets was significantly higher than that of fish fed the CON diet. The lysozyme activity of fish fed the PT, GRO, GROMW, GROPT and GROTW diets was significantly higher than that of fish fed the CON and OTC diets. The myeloperoxidase activity of fish fed the TW, GRO, GROMW, GROPT and GROTW diets was significantly higher than that of fish fed the CON, PT and MW diets (p < 0.05). The flounder growth hormone gene expression of fish fed the TW, GRO, GROMW, GROPT, GROTW and OTC diets was significantly higher than that of fish fed the CON, PT and MW diets (p < 0.05). The interleukin 1ß and interleukin 10 gene expressions of fish fed the GRO, GROMW, GROPT and GROTW diets were significantly higher than those of fish fed the CON, PT, TW and MW diets (p < 0.05). Intestinal histology showed a significantly higher villi length for fish fed the GRO, GROMW, GROPT and GROTW diets compared to that of fish fed the CON diet (p < 0.05). Digestive enzyme activities such as trypsin activity were significantly higher in fish fed the GROMW, GROPT and GROTW diets than those in the rest of the diet groups (p < 0.05). Amylase activity in fish fed the MW, GRO, GROMW, GROPT, GROTW and OTC diets was significantly higher than that of fish fed the PT, TW and CON diets (p < 0.05). On the other hand, the lipase activity of fish fed the TW, GRO, GROMW, GROPT and GROTW diets was significantly higher than that of fish fed the CON, PT, MW and OTC diets (p < 0.05). The cumulative survival rate of fish fed the PT, GROTW, GROPT and GROMW diets was significantly higher than that of fish fed the CON, TW and MW diets after thirteen days of the challenge testing. Overall, the results demonstrate that the GRO, GROMW, GROPT and GROTW diets could be beneficial feed additives to replace antibiotics in juvenile olive flounder fed low-fish-meal diets.

Investigation of Gene Networks in Three Components of Immune System Provides Novel Insights into Immune Response Mechanisms against Edwardsiella tarda Infection in Paralichthys olivaceus.

As a quintessential marine teleost, Paralichthys olivaceus demonstrates vulnerability to a range of pathogens. Long-term infection with Edwardsiella tarda significantly inhibits fish growth and even induces death. Gills, blood, and kidneys, pivotal components of the immune system in teleosts, elicit vital regulatory roles in immune response processes including immune cell differentiation, diseased cell clearance, and other immunity-related mechanisms. This study entailed infecting P. olivaceus with E. tarda for 48 h and examining transcriptome data from the three components at 0, 8, and 48 h post-infection employing weighted gene co-expression network analysis (WGCNA) and protein-protein interaction (PPI) network analysis. Network analyses revealed a series of immune response processes after infection and identified multiple key modules and key, core, and hub genes including xpo1, src, tlr13, stat1, and mefv. By innovatively amalgamating WGCNA and PPI network methodologies, our investigation facilitated an in-depth examination of immune response mechanisms within three significant P. olivaceus components post-E. tarda infection. Our results provided valuable genetic resources for understanding immunity in P. olivaceus immune-related components and assisted us in further exploring the molecular mechanisms of E. tarda infection in teleosts.