Paralichthys Olivaceus Research Articles

Raw Fish-based Moist Pellet and Extruded Pellet on Growth, Levels and Expression of GH/IGF Axis, Intestinal Enzyme Activity and Gastrointestinal Morphology of Olive Flounder (Paralichthys olivaceus): An Actual Field Study

A six-month feeding trial was conducted to investigate the effect of moist pellet (MP) and commercial extruded pellet (EP) on the somatic growth, level and expression of GH/IGF axis, intestinal enzyme activity and gastrointestinal morphology of olive flounder (Paralichthys olivaceus) reared under actual farm conditions. MP was a raw fish-based diet composed of mackerel and cutlass fish while EP diet was a locally-produced commercial feed. Weight growth rate showed no significant difference (P>0.05), although EP-fed fish had a better somatic yield. Feed conversion ratio was better and condition factor has significantly improved when given EP diet (P<0.05). Plasma level of growth hormone and insulin-like growth factor 1 was comparably similar between diets, but EP-fed olive flounder had a higher hepatic IGF-1 expression (P<0.05). The activity of trypsin, chymotrypsin and lipase were insignificantly influenced by diets (P>0.05). The overall gastrointestinal morphology was in similitude regardless of diets, but EP- fed fish had a longer intestinal villi length and goblet cell count in the pyloric caeca (P<0.05). These dietary induced differences are discussed to have partly promoted better growth in EP diet. The findings suggest an equivalent, or better utilization of EP diet in the actual farm operation.

Establishment and characterization of an intestinal epithelial cell line from Japanese flounder (Paralichthys olivaceus)

The Japanese flounder Paralichthys olivaceus is a high-value mariculture species. However, the aquaculture industry worldwide faces significant challenges due to flounder ascites, a serious disease caused by gram-negative bacterium Edwardsiella infection of fish cultures. Studies have confirmed that Edwardsiella tarda infection of flounder causes intestinal inflammatory disease. The recognition of pathogenic microorganisms by intestinal epithelial cells is the primary cause of inflammatory responses. In this study, a new cell line derived from the distal intestine (mainly the mid and posterior intestine) of Japanese flounder was established and characterized. This cell line, designated as the PoIEC line, was maintained in DMEM with 15% fetal bovine serum at 24 °C for >1 year, and subcultured >55 times. The PoIECs were epithelial-like cells with cobblestone-like shapes. Microvilli were observed on the surface of the PoIECs through transmission electron microscopy and the existence of epithelial markers was confirmed. PoIECs transfected with a pEGFP-N3 plasmid emitted an intense green fluorescence signal. Transfection efficiency of these cells was calculated to be approximately 37%, making this cell line a potential tool for future exogenous gene function research. Furthermore, PoIECs were stimulated by LPS and live E. tarda, and the expression profiles of immune-related genes (MHC Iα, MHC IIα, TNFα, IL-8, and IL-1β) confirmed that these cells recognized antigens and activated immune responses. In conclusion, a new cell line derived from the intestine of Japanese flounder was established, and this cell line was an ideal cellular platform for the study of mucosal immunity, probiotic flora and nutrient metabolism in the fish gut.

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.

Disposable microRNA biosensors based on dual-role polymer-dispersed silver nanowires for diagnosis of viral hemorrhagic septicemia virus infection in olive flounder

Fish farms often suffer from viral infections that can lead to mass mortality. Early detection of these infections can help prevent the spread of the infection. microRNA (miRNA) is a new biomarker for detecting early infections in farmed olive flounder. We developed a disposable miRNA biosensor by utilizing screen-printed carbon electrodes with polymer-dispersed silver nanowires (P-AgNWs). The target miRNA here was miR-15b-5p, which is considered a specific candidate for detecting viral hemorrhagic septicemia virus (VHSV) infection in fish. A capture probe (CP), a thiol-modified DNA oligonucleotide chain complementary to the miR-15b-5p, was immobilized on the surface of P-AgNWs. While the hybridization of miR-15b-5p with the CP is monitored using square wave voltammetry techniques based on the specific binding of methylene blue (MB) to the DNA-RNA hybrid formed between CP and the miR-15b-5p. The miRNA biosensor demonstrated a wide linear response range of 1.0–100 fM under optimum conditions with a low detection limit of 0.70 fM. According to a series of voltammetric experiments, the approach demonstrated acceptable precision and sensitivity for determining the miR-15b-5p levels in olive flounder infected with VHSV.

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.

Ecosystem-based fishery management that accounts for trade-offs between fishing and enhancement stocking: A case study of Juehua Island in the Bohai Sea

The implementation of marine ecosystem management requires modeling that accounts for the trade-off between exploitation in fisheries and protection activities (e.g., enhancement and release, artificial reefs). Researchers are increasingly interested in the trends of fishery resources under these joint driving forces due to the negative impacts of resource exploitation and the positive impacts of marine protection. Using the sea around Juehua Island in China’s Bohai Sea as a case study, we developed a marine ecosystem model based on the Ecopath with Ecosim software to analyze the trends in fishery resources driven both by fishing and stock enhancement. When the fishing effort exceeded 42 % of the 2019 value, the biomass of top predator Platycephalus indicus decreased by 50 %, indicating a regime shift. When the fishing effort increased to 340 % of the 2019 level, the ecosystem shifts to an alternative stable state occurs, indicating destabilization of the marine ecosystem. The impact of fishing effort on the marine ecosystem was not well compensated by release of a single target species. We also designed an orthogonal experiment with three factors (the key species Fenneropenaeus chinensis, Paralichthys olivaceus, and Charybdis japonica) and four restocking release levels to determine both the optimal release rate for each species individually and the optimal combination of different species. If the 2019 fishing effort is maintained, the release of 125 × 106 juvenile F. chinensis, 400 × 103 juvenile P. olivaceus, and 15 × 106 juvenile C. japonica would be an optimal solution to improve the marine biomass, trophic level, and biodiversity. Our results provide insights into ecosystem-based management strategies for implementing a policy to limit fishing in the study area and for scientifically determining the restocking release rates.

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.

Pharmacokinetic-Pharmacodynamic Analysis of Cephalexin against Streptococcus parauberis in Olive Flounder (Paralichthys olivaceus: Temminck and Schlegel)

Olive flounders are susceptible to annual outbreaks of streptococcosis, which accounts for approximately 10% of all fish farm diseases and is associated with high mortality rates. The development of an antibiotic therapy against streptococcosis is thus necessary. This study determined the therapeutic effects of varying cephalexin concentrations in Streptococcus parauberis-infected olive flounders and evaluated its histopathological toxicity and residual concentration in the fish. Compared with the control group, the 200 and 800 mg/kg cephalexin groups showed significant mean survival rates of approximately 10% and 30%, respectively, and the 400 mg/kg group showed the highest survival rate of approximately 40%. The average residual cephalexin concentration in muscle samples on day 1 post-cephalexin administration was 13.21 µg/kg, showing a rapid decrease. At the optimum water temperature (25°C), cephalexin was rapidly metabolized within 24 hr of its administration being terminated, and most of it was excreted from the bodies of the fish. A histopathological analysis showed that the oral administration of cephalexin did not lead to specific inflammatory lesions, and there were no significant differences between the treatment and control groups. Our novel findings suggest that cephalexin is a promising candidate for treating streptococcosis outbreaks in fish farms.

The persistence of viral hemorrhagic septicemia virus (VHSV) mRNA in juvenile olive flounder, Paralichthys olivaceus following live VHSV vaccination

Several studies have been conducted on vaccines for viral hemorrhagic septicemia virus (VHSV) in order to prevent VHSV infection. Effective live VHSV vaccines have been reported for suboptimal temperatures (17–20 °C). Previous strategies for live vaccine immunization periods required more than two weeks for fish to acquire resistance. In our study, we demonstrate the efficacy of a 10-day live VHSV vaccine immunization period at 17 °C. We also investigated the presence of viral mRNA genes in vaccinated juvenile olive flounder after challenge at 10 °C using RNA in situ hybridization (ISH) and quantitative polymerase chain reaction (qPCR). The live VHSV-vaccinated flounder exhibited a 100% survival rate for 20 days post-challenge (dpc) at 10 °C, whereas all non-vaccinated flounder had perished by 13 dpc. Viral mRNA was detected in the spleen and heart tissue of vaccinated flounder through RNA ISH visualization, whereas no signal was observed in the kidney. The viral mRNA copy number in internal organs (heart, kidney, and spleen) of vaccinated flounder was lower than that in non-vaccinated flounder. These findings support the effectiveness of a 10-day rearing period following live VHSV vaccination and suggest its potential for shortening the VHSV vaccine program.

Novel anti-atrophic peptide isolated from olive flounder surimi as a nutraceutical additive against TNF-α induced muscle atrophy

Skeletal muscle atrophy has significant negative functional effects, especially on patients with inflammatory diseases. Tumor necrosis factor alpha (TNF-α) plays a crucial role in muscle pathology associated with muscle wasting and impairment of differentiation. The present study evaluated the anti-atrophic activity and related mechanism of a novel peptide (Tryptophan-Tyrosine-Lysine) derived from Olive Flounder surimi (OFSP), against TNF-α induced muscle atrophy in C2C12 myotubes. Molecular docking results showed that OFSP binds to TNF-α, inhibiting its binding to TNFR1 receptor. OFSP treatment increased the cell viability and decreased intracellular reactive oxygen species production caused by TNF-α. OFSP markedly downregulated the NF- κB pathway proteins phosphorylation, thereby inhibiting pathway activation. Furthermore, OFSP suppressed IL-6 production and expression of E2 ubiquitin ligases, atrogin-1/MAFbx and MuRF1, while increasing myogenic marker expressions. Current findings indicate that OFSP exhibits excellent anti-atrophic activity against inflammation-induced muscle atrophy, and these characteristics may be valuable in anti-atrophic functional food development.

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-1β, 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.

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.

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.

Development of a trivalent vaccine for prevention of co-infection by Miamiensis avidus and Tenacibaculum maritimum in farmed olive flounder

Development of a trivalent vaccine for prevention of co-infection by Miamiensis avidus and Tenacibaculum maritimum in farmed olive flounder

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-1β, 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.

The uninhibited glucagon signaling pathway in Japanese flounder Paralichthys olivaceus: A link with the glucose intolerance?

The study aimed to explore the role of glucagon in regulating glucose metabolism and gain insight into the reasons for glucose intolerance in carnivorous fish. Firstly, Japanese flounder was injected intraperitoneally with glucagon and small interfering RNA (siRNA) targeted glucagon receptor a (GCGRa), cAMP-response element binding protein 1 (CREB1), and peroxisome proliferator-activated receptor-γ-coactivator 1α (PGC-1α). Secondly, a two-week feeding trial was conducted with Japanese flounder fed diets with 11.95% and 31.96% dietary carbohydrates, respectively. At the start and 7th day of the feeding trial, flounders fed each diet were intraperitoneally injected with saline or siGCGRa. Furthermore, the fish were separated into two groups and fasted for 72 h. Saline and siGCGRa were injected respectively at 6 h after feeding. The results showed that glucagon activated gluconeogenesis in the liver, leading to an elevation of plasma glucose levels, while interference of GCGRa, CREB1, and PGC-1α caused the opposite results. Elevated dietary carbohydrate intake increased plasma glucose and insulin levels. Meanwhile, the glucagon signaling pathway was activated. After interference with the GCGRa, plasma glucose decreased, and the glucagon pathway was inhibited. The plasma insulin increased, and the mRNA expression of protein kinase B β (akt2) was up-regulated accordingly. In addition, upon injecting siGCGRa, it was observed that gluconeogenesis in the liver and plasma glucose levels during fasting were reduced in comparison to the control group. In contrast, plasma insulin levels and the transcription levels of akt2 and glucokinase (gck) in the liver exhibited an increase. In summary, the uninhibited glucagon signaling pathway promoted gluconeogenesis and suppressed the action of insulin in Japanese flounder, which might be the reason for the glucose intolerance.

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.

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.

Accurate genomic selection using low-density SNP panels preselected by maximum likelihood estimation

Genomic selection (GS) poses a challenge for the prediction of the genomic estimated breeding value (GEBV) using a low-density SNP panel. Several methods have been proposed for SNP preselection. However, these methods often suffer from either significant computational complexity or erratic accuracy in GS. In this study, we developed an approach called MLE-rank based on maximum likelihood estimation to preselect a set of SNPs for GS. First, we generated 90 simulated datasets and compared the performance of MLE-rank with uniform distribution and preselection based on a genome-wide association study (GWAS). For simulated datasets, compared to uniform distribution, both MLE-rank and GWAS preselection reduced the SNP density by a factor of 10 while maintaining prediction accuracy. Additionally, compared to the other two methods, MLE-rank's prediction accuracy was significantly improved with the medium- and high-heritability datasets. Then, we further evaluated these three preselection approaches using real disease-resistant phenotypes of leopard coral grouper (Plectropomus leopardus) and Japanese flounder (Paralichthys olivaceus). We found that the 3 k SNPs preselected by MLE-rank had a stable and effective prediction effect. The uniform distribution requires 70 k, while the GWAS preselection method requires 3 k (P. leopardus) and 50 k (P. olivaceus) to achieve similar prediction accuracy. Finally, we evaluated the prediction accuracy of MLE-rank using candidate populations of flounders and their progeny survival rates, with uniform distribution and GWAS preselection as benchmarks. In the results for this dataset, MLE-rank was found to have the same predictive effect for low-density SNP panels as it did for high-density SNPs, regardless of whether GWAS preselection or uniform distribution was used. Taken together, the results we have observed indicate that we have ensured that MLE-rank does not reduce prediction accuracy for any of the datasets. MLE-rank showed superior performance in reducing the number of SNPs. Moreover, we observed a relative standard deviation in prediction accuracy when using a low density of SNPs selected by MLE-rank compared to a high density determined through a uniform distribution strategy. In conclusion, MLE-rank not only reduces the number of SNPs used for GS but also exhibits high predictive accuracy. This could potentially lead to a decrease in genotyping costs and promote the wider application of GS in fish breeding.

Functional characterization of obscure puffer ToNK-lysin: A novel immunomodulator possessing anti-bacterial and anti-inflammatory properties

NK-lysins are one of the most abundant antimicrobial peptides produced by cytotoxic T lymphocytes (CTLs) and natural killer cells (NKs), and identified as a new class of intrinsically disordered proteins, playing critical roles in the cell-mediated cytotoxicity response, as well as immunomodulatory and antimicrobial activities upon a significant range of pathogens. In the present study, an NK-lysin was identified from Obscure puffer Takifugu obscurus (ToNK-lysin). The open reading frame of ToNK-lysin sequence spans 423 bp, encoding a peptide with 140 amino acids which shares a moderate residue identity (18%–60%) with NK-lysin of mammals and other teleost species. Phylogenetic analysis revealed that ToNK-lysin was most closely related to NK-lysins from the Pleuronectiformes (Bastard halibut Paralichthys olivaceus and Pacific halibut Hippoglossus stenolepis). Comprehensive computational analysis revealed that ToNK-lysin have substantial level of intrinsic disorder, which might be contribute to its multifunction. The transcripts of the ToNK-lysin were detected in multiple examined tissues and most abundant in gills. After bacterial and Poly I:C challenge, the transcriptional levels of ToNK-lysin were significantly up-regulated in the head kidney, liver and spleen at different time points. The recombinant ToNK-lysin showed significant antibacterial activity against Vibrio harveyi and Escherichia coli, and the ToNK-lysin treatment not only reduced the bacterial loads in liver and head kidney, but also alleviated the pathogen-mediated upregulation of immune-related genes. In addition, the co-incubation with rToNK-lysin protein remarkably degraded bacterial genomic DNA, suggesting the potential mechanism of ToNK-lysin against microbes. These results suggest that ToNK-lysin possess antibacterial and immunoregulatory function both in vivo and in vitro, which may allow it a potential applicability to the aquaculture industry.