Genome-wide identification, phylogenetic relationships and expression patterns of the NOD-like receptor (NLR) gene family in flounder (Paralichthys olivaceus).

NOD-like receptors (NLRs) are one of the pattern recognition receptors which have been widely known for identifying pathogens and regulating innate immunity in mammals, but the functions of the NLR gene family in teleost fish remain poorly understood. In this study, we conducted a comprehensive identification and analysis of the flounder (Paralichthys olivaceus) NLR gene family, including bioinformatics information, evolutionary relationships, gene structures, conserved motifs, domain composition, expression patterns and protein-protein interaction (PPI). We identified 22 NLRs in flounder (flNLRs) which were clustered into three subfamilies according to their domain organizations and phylogenetic features, i.e., NLR-A (6 members) resembling mammalian NODs, NLR-B (1 member) resembling mammalian NLRPs, and NLR-C (15 members) unique to teleost fish. All flNLRs shared a conserved NACHT domain including an N-terminal nucleotide-binding domain, a middle helical domain 1, and a winged helix domain. Gene structure analysis displayed that flNLRs were significantly different, with exon numbers from 1 to 52. Conserved domain analysis showed that the N-terminus of flNLRs possessed different characteristics of the domains including CARD domain, PYRIN domain, RING domain, and fish-specific FISNA domain, and the C-terminus of seven NLR-C members contained an extra B30.2 domain, named NLRC-B30.2 group. Notably, flNLRs were expressed in all nine tested tissues, showing higher expressions in the systemic and mucosal immune tissues (e.g., kidney, spleen, hindgut, gills, skin, liver) in healthy flounder, and significant responses to intraperitoneal injection and immersion immunization of inactivated Vibrio anguillarum in mucosal tissues, especially the NLR-C members. In addition, PPI analysis demonstrated that some flNLRs of NLR-A and NLR-C shared the same interacting proteins such as RIPK2, TRAF6, MAVS, CASP, ASC, and ATG5, suggesting they might play crucial roles in host defense, antiviral innate immunity, inflammation, apoptosis and autophagy. This study for the first time characterized the NLR gene family of flounder at the genome-wide level, and the results provided a better understanding of the evolution of the NLR gene family and their immune functions in innate immunity in fish.

Identification of rearing temperature-dependent host defense signaling against viral hemorrhagic septicemia virus infection.

Viral hemorrhagic septicemia virus (VHSV) infection is associated with fatal outcomes in the aquaculture production of olive flounder (Paralichthys olivaceus). Olive flounders at low and high temperatures are known to be highly susceptible and resistant to VHSV infection, respectively. To study temperature-dependent innate immune activity, 4-aminobenzoic hydrazide (4-AH), a myeloperoxidase (MPO) inhibitor, was used to treat VHSV-infected olive flounders reared at a high temperature of 20 °C (20VI). Mortality, the MPO transcription, and the proteomic expression pattern of the 20VI group were then compared with those of groups of VHSV-infected flounders reared at 15 °C (15V) and 20 °C (20V). The cumulative mortality rate of the 20VI group was increased by 35% compared with that of the untreated 20V group. The MPO transcription was decreased 5.8-fold in 20VI than in 20V group. Its expression decreased further at a lower temperature and after exposure to VHSV. Histopathological analysis revealed necrosis of splenic tissue in 20VI and 15V, but not in 20V group. Based on clustering analysis, proteins with increased expression in 15V and 20VI groups were associated with viral mRNA translation and reproduction compared with those of 20V group. Increased expression of DHX58, MX1, and UBB was detected in 15V and 20VI groups, suggesting a role in triggering innate immune response. Unfortunately, these genes failed to induce the translocation of GLUT4 to the surface membrane from the intracellular location due to decreased expression of 14-3-3 proteins (YWHAB and YWHAZ) and microtubules (TUBA1A and TUBB4B). Suppression of glucose supply led to inactivation of MPO and suppression of MHC-I and MHC-II-linked immune activity, resulting in high viral infection and spread. In conclusion, this study highlights that defective GLUT4 translocation-dependent glucose uptake increases the mortality of VHSV-infected olive flounders by inhibiting MPO activity.

Inhibition of lysozyme lytic activity by Ivy derived from Photobacterium damselae subsp. piscicida.

A pseudotuberculosis pathogen, Photobacterium damselae subsp. piscicida (Pdp), has caused enormous economic damage to yellowtail aquaculture in Japan. The Ivy gene has been discovered in plasmid of Pdp, and it has been proposed that it may help bacteria evade lysozyme-mediated lysis during interaction with an animal host. However, the lysozyme-inhibiting activity of Pdp-derived Ivy (Ivy-Pdp) is unknown, and it is unclear whether it acts as a virulence factor for host biophylaxis. In this study, the inhibitory effect of Ivy-Pdp on lysozyme was evaluated by expressing and purifying the recombinant Ivy-Pdp protein (rIvy-Pdp). The rIvy-Pdp protein inhibited hen egg white lysozyme activity in an rIvy-Pdp-concentration-dependent manner, and its inhibitory effect was similar under different temperature and pH conditions. The serum and skin mucus of the yellowtail (which is the host species of Pdp), Japanese flounder, and Nile tilapia showed bacteriolytic activity. In contrast, the addition of rIvy-Pdp inhibited the lytic activity in the serum of these fish species. In particular, it significantly inhibited lytic activity in the serum and skin mucus of Nile tilapia. On the basis of these results, we suggest that Ivy-Pdp is a temperature- and pH-stable lysozyme inhibitor. Additionally, Ivy-Pdp inhibited the lytic activity of lysozyme, which is involved in host biophylaxis. In summary, we inferred that Ivy-Pdp is an important factor that diminishes the sterilization ability of C-type lysozyme when Pdp infects the host.

Peripheral Blood B-Lymphocytes Are Involved in Lymphocystis Disease Virus Infection in Flounder (Paralichthys olivaceus) via Cellular Receptor-Mediated Mechanism.

Previous studies imply that peripheral blood leukocytes (PBLs) may play an important role in systemic lymphocystis disease virus (LCDV) dissemination, but whether the PBLs are susceptible and permissive to LCDV infection and the dissemination mechanism need to be clarified. In this study, LCDV was firstly confirmed to infect the PBLs in flounder (Paralichthys olivaceus) in vivo, and to replicate in PBLs in vitro. Subsequently, the 27.8 kDa receptor protein (27.8R), a functional receptor mediating LCDV infection in flounder gill cells, was shown to locate on the cell membrane of PBLs and co-localize with LCDV in PBLs, while blocking of the 27.8R via pre-incubation of anti-27.8R MAb with the PBLs could obviously inhibit LCDV infection, revealing the 27.8R as a receptor for LCDV entry into PBLs. Multicolor fluorescence imaging studies verified that IgM+ and IgD+ B-lymphocyte were involved in LCDV infection. In the sorted IgM+ B-cells, 27.8R+ and LCDV+ signals were simultaneously observed, and LCDV copy numbers increased with time, indicating that IgM+ B-cells expressed the 27.8R and were permissive to LCDV infection. Furthermore, the dynamic changes of IgM+, 27.8R+, LCDV+ and LCDV+/IgM+ PBLs were monitored during the early phase of LCDV infection. It was found that the percentage of IgM+ B-cells in PBLs clearly declined first and then increased, suggesting LCDV infection facilitated damage to B-cells, whereas the amounts of 27.8R+ and LCDV+ PBLs, as well as LCDV-infected IgM+ B-cells, showed an opposite trend. These results proved that IgM+ B-lymphocytes could be infected by LCDV via a receptor-mediated mechanism and support viral replication, which provided novel insights for the first time into the role of B-lymphocytes in LCDV dissemination and pathogenesis in teleost fish.

Molecular cloning and characterization of chemokine C-C motif ligand 34 (CCL34) genes from olive flounder (Paralichthys olivaceus).

Chemokines are a superfamily of chemotactic cytokines that regulate the migration and immune responses of leukocytes. Depending on the arrangement of the first two cysteine residues, chemokines are divided into four groups: CXC (α), CC (ß), C (γ), and CX3C (δ). Chemokine C-C motif ligand 34 (CCL34) is a member of the CC chemokine family and is known as a fish-specific CC chemokine. In this experiment, we analyzed the molecular cloning and characterization of the PoCCL34 gene in olive flounder (Paralichthys olivaceus), including CCL34a.3 (PoCCL34a.3) and CCL34b.3 (PoCCL34b.3). The amino acid sequence of PoCCL34 has four highly conserved cysteine residues and it has a C-C motif. Phylogenetic analysis revealed that PoCCL34 was phylogenetically clustered in the fish CCL34 subcluster. Recombinant PoCCL34 induced chemotaxis of head kidney leukocytes in a dose-dependent manner. Head kidney leukocytes stimulated with PoCCL34 also exhibited significant respiratory burst activity and increased expression of pro-inflammatory cytokines (IL-1ß, IL-6, and CXCL8), but the overall expression of interferon-related genes (IFN-α/ß, IFN-γ, Mx, and ISG15) did not increase. Olive flounder injected with recombinant PoCCL34 demonstrated increased expression of pro-inflammatory cytokines (IL-1ß and IL-6) in the head kidney. However, there was no increase in the expression of interferon-related genes (IFN-α/ß, IFN-γ, Mx, and ISG15). Additionally, recombinant PoCCL34 induced high lysozyme activity in the serum of the flounder. These results indicate that although PoCCL34 is not involved in the antiviral response, it may play a significant role in the overall immune response of the flounder, particularly in mediating the inflammatory response.

Role of Endogenous Cathepsin L in Muscle Protein Degradation in Olive Flounder (Paralichthys olivaceus) Surimi Gel.

We investigated the effect of endogenous cathepsin L on surimi gel produced from olive flounder (Paralichthys olivaceus). The amino acid sequences of six proteins predicted or identified as cathepsin L were obtained from the olive flounder genome database, and a phylogenetic analysis was conducted. Next, cathepsin L activity toward N-α-benzyloxycarbonyl-l-phenylalanyl-l-arginine-(7-amino-4-methylcoumarin) (Z-F-R-AMC) was detected in crude olive flounder extract and a crude enzyme preparation. A considerable decrease in the level of myosin heavy chain (MHC) in surimi occurred during autolysis at 60 °C. In contrast, the levels of actin, troponin-T, and tropomyosin decreased only slightly. To prevent protein degradation by cathepsin L, a protease inhibitor was added to surimi. In the presence of 1.0% protease inhibitor, the autolysis of olive flounder surimi at 60 °C was inhibited by 12.2%; the degree of inhibition increased to 44.2% as the inhibitor concentration increased to 3.0%. In addition, the deformation and hardness of modori gel increased as the inhibitor concentration increased to 2.0%. Therefore, cathepsin L plays an important role in protein degradation in surimi, and the quality of surimi gel could be enhanced by inhibiting its activity.

Sexual dimorphism of DNA and histone methylation profiles in the gonads of the olive flounder Paralichthys olivaceus.

DNA methylation and histone methylation are two types of the most important epigenetic modifications. However, research on their differential expression in gonads of male and female fish is limited. In this study, we examined the characteristics of DNA methylation and tri-methylation of lysine 4 of histone H3 (H3K4me3) modification profiles in the gonads of the wild-type and meio-gynogenetic olive flounders Paralichthys olivaceus. Enzyme-linked immunosorbent assay (ELISA) analysis revealed that the global DNA methylation level was higher in the testis than in the ovary. Real-time quantitative PCR (qPCR) results indicated that maintenance DNA methyltransferase gene dnmt1 and de novo DNA methyltransferase gene dnmt3a are highly expressed in the ovary, while DNA demethyltransferase genes tets are highly expressed in the testis. The inconsistency of DNA methylation and methyltransferase genes in the gonads might associate with the differential distribution in the testis. 5-mC mainly located in the spermatids of the testis was shown with immunohistochemistry (IHC). Furtherly, dnmt3a and tets are mainly located in spermatocytes and oocytes with in situ hybridization (ISH) analysis. As for H3K4me3, total level is higher in the ovary detected with western blot assay. IHC results showed that the signals of H3K4me3 in Sertoli cells of the testis were stronger than those in spermatocytes and spermatids. Methyltransferase gene kmt2b and demethylase genes kdm5a and kdm5c also exhibit much higher expression in the testis with qPCR, and ISH stronger signals of kmt2b and kdm5s were detected in spermatocytes. These results implied that DNA methylation and H3K4me3 are involved in the flounder sex differences and gametogenesis.

Analyses of mRNA-seq and miRNA-seq of the brain reveal the sex differences of gene expression and regulation before and during gonadal differentiation in 17ß-estradiol or 17α-methyltestosterone-induced olive flounder (Paralichthys olivaceus).

Olive flounder (Paralichthys olivaceus) is a commercially important flatfish species cultured in East Asia. Female flounders generally grow more rapidly than males, therefore control of the sex ratio seems to be a proposed way to increase production. However, the sex determination gene and sex determination mechanism have yet been elucidated. The brain is an important organ that is involved in gonadal development. To explore the sex differences of gene expression in the brain before and during the flounder gonadal differentiation, we used messenger RNA (mRNA)-seq technology to investigate transcriptomes of male and female brains. Between female and male brains, 103 genes were differentially expressed before ovarian differentiation, 16 genes were differentially expressed before testicular differentiation, and 64 genes were differentially expressed during gonadal differentiation. According to annotation and Kyoto Encyclopedia of Genes and Genomes information, the differentially expressed genes (DEGs) were involved in circadian rhythm, circadian rhythm-fly, circadian entrainment, dopaminergic synapse, calcium signaling, glutamatergic synapse, taste transduction, herpes simplex infection, long-term depression, retrograde endocannabinoid signaling, and the synaptic vesicle cycle pathways. MicroRNA (miRNA)-seq was performed during the gonadal differentiation and the target genes of miRNAs were predicted. Integrated analysis of mRNA-seq and miRNA-seq showed that 29 of the 64 DEGs were regulated by the differentially expressed miRNAs during the gonadal differentiation. Our study provides a basis for further studies of brain sex differentiation and the molecular mechanism of sex determination in olive flounder.

Current use and management of commercial fish vaccines in Korea.

The aquaculture industry in Korea has grown rapidly since the 1960s, and it is a major food source. However, the expansion of aquaculture systems has increased the chances of infectious disease outbreaks, and vaccination plays an important role in commercial fish farming. This is the first comprehensive review of commercial fish vaccines in Korea. It not only provides an overview of commercially available fish vaccines and their associated approval processes and laws, but also some perspectives on research advances regarding fish vaccines in Korea. In Korea, fish vaccines are approved only after their safety and effectiveness have been verified according to the Pharmaceutical Affairs Act, and after approval, each vaccine lot must pass the national evaluation criteria. As of the end of 2019, 29 vaccines were approved for 10 fish pathogens, including both single and combination vaccines containing more than two inactivated pathogens. The approved fish vaccines consist of 2 immersion vaccines, as well as 1 intramuscular and 26 intraperitoneal vaccines, which require syringe injection. All the 29 vaccines are manufactured as formalin-inactivated vaccines; 1 is an adjuvant vaccine and 28 are non-adjuvant vaccines; 25 are bacterial vaccines, 2 are viral vaccines, 1 is a parasite vaccine, and 1 is a parasite and bacterial vaccine. In terms of the target fish species, 27 vaccines are used in the olive flounder (Paralichthys olivaceus), 1 in the starry flounder (Platichthys stellatus), and 1 in the red seabream (Pagrus major), striped beakfish (Oplegnathus fasciatus), and amberjack (Seriola quinqueradiata). This imbalance exists mostly because the olive flounder is the main farmed fish species in Korea. In 2018, 67.71 million vaccine doses were distributed following satisfactory performance in the national evaluation. They were used to vaccinate approximately 80.6% of farmed olive flounders.

Dynamic distribution of formalin-inactivated Edwardsiella tarda in flounder (Paralichthys olivaceus) post intraperitoneal vaccination.

In order to investigate the dynamic distribution of antigen in different tissues post vaccination, an absolute real-time quantitative PCR was employed to detect the amount of antigen in flounder (Paralichthys olivaceus) post intraperitoneal (i.p.) injection with three concentrations (107, 108, 109 CFU ml-1) of formalin-inactivated Edwardsiella tarda bacterin. The results showed that the amount of uptaken antigen quickly increased and then decreased in different tissues. The peak occurred first in the spleen and head kidney at 6-9 h after injection, and in the liver and blood at 9-15 h, then in the gill, intestine and skin at 15-24 h, finally in the muscle at 24-36 h. The amount of antigen was highest in the spleen and head kidney, followed by the blood, liver and gill, and lowest in the intestine, skin and muscle. Among the three concentration groups, the amount of antigen increased with the increasing concentration of the vaccine in the blood, liver, gill, intestine, skin and muscle, except for the spleen and head kidney, in which more antigens were found in the 108 CFU ml-1 group than that in 109 CFU ml-1 group. Moreover, IIFA and western blotting was performed to examine the tissue distribution of antigen at 9 h after vaccination with 108 CFU ml-1 formalin-inactivated E. tarda. The bacteria were mainly observed in the spleen and head kidney, then the liver, gill and blood, and least in the intestine, skin and muscle, which was roughly in accordance with the results of absolute qPCR. Furthermore, the expressions of CD4-1, MHC IIα, CD8α and MHC Iα in different tissues were detected by RT-qPCR, and the expression levels of these genes were highest in the spleen and head kidney, then in the blood, gill, liver, and lowest in the intestine, skin and muscle. All these results provided useful information for dynamic transportation of antigen uptake post vaccination, and also deepened the understanding of immune response to the injection vaccination.

Interleukin-2 (IL-2) in flounder (Paralichthys olivaceus): Molecular cloning, characterization and bioactivity analysis.

Interleukin-2 (IL-2) is mainly produced by CD4+ T helper lymphocytes, which is an important immunomodulatory cytokine that primarily promotes activation, proliferation and differentiation of T cells. In the present study, flounder (Paralichthys olivaceus) interleukin 2 homologue (poIL-2) was identified for the first time, and its expression patterns were characterized in healthy, virus- or bacteria-infected flounder. The full-length cDNA sequences of poIL-2 was 989 bp with an open reading frame of 423 bp coding a polypeptide of 140 amino acids (aa). The deduced aa sequences shared low similarities (<53%) with other known fish IL-2s. Multiple alignment of aa sequences revealed that poIL-2 own the classical IL-2 family signature of "C-X(3)-EL-X(2)-(T/V)-(V/M/L)-(K/T/R)-X-EC" and "DS-X-(F/L)Y(A/T/S)P". In healthy flounder, IL-2 mRNA was highly expressed in PBLs, spleen and hindgut, and moderately expressed in gill, trunk kidney and stomach. PHA, LPS and Con-A could effectively induce poIL-2 expression in primary cultured peripheral blood leukocytes in vitro. poIL-2 transcripts were significantly up-regulated in spleen, kidney, gill and hindgut post infections with Edwardsiella tarda and Hirame novirhabdovirus (HIRRV). The eukaryotic expression vector encoding poIL-2 (pcIL-2) was constructed and intramuscularly injected, which could be successfully expressed in flounders and induced significantly higher expressions of six immune related genes including poIL-2, ß-defensin, CD4-1, CD8α, IFN-γ and TNF-α compared with the injection with control plasmid. Moreover, pretreatment with pcIL-2 could markedly increase the survival rate of flounder challenged with HIRRV. Our results demonstrated that poIL-2 plays an important role in the induction of immune responses and immune defense against bacterial and virus infection, which indicated its potential use as an immunopotentiator to prevent diseases in flounder.

Nonconservation of TLR5 activation site in Edwardsiella tarda flagellin decreases expression of interleukin-1ß and NF-κB genes in Japanese flounder, Paralichthys olivaceus.

Flagellin is the subunit protein that composes bacterial flagella and is recognized by toll-like receptor 5 (TLR5) as a ligand. Flagellin protein (e.g., FliC and FlaA) contains the D1, D2, and D3 domains; the D1 domain is important for recognition by TLR5 for activation of the innate immune system. In teleosts, there are two types of TLR5, the membrane form (TLR5M) and soluble form (TLR5S), the latter of which is not present in mammals. In this study, the potential of flagellin from Edwardsiella tarda (EtFliC) to induce inflammation-related genes interleukin (IL)-1ß and NF-κB-p65 through TLR5S in Japanese flounder (Paralichthys olivaceus) was elucidated. A transient overexpression system was developed in flounder natural embryonic (HINAE) cells using constructs encoding two flagellin genes derived from E. tarda (pEtFliC) and Escherichia coli (pEcoFliC) and the flounder TLR5S gene (pPoTLR5S). Expression of inflammation-related genes in EtFliC- and PoTLR5S-overexpressing HINAE cells was significantly lower than in EcoFliC- and PoTLR5S-overexpressing cells. To clarify the difference between EtFliC and EcoFliC potency, the amino acid sequence of EtFliC was compared with that of other bacterial flagellin. The 91st arginine residue, known as the mammalian TLR5 activation site, was conserved in the flagellin of E. coli and other bacteria but not in EtFliC. To reveal the importance of the 91st arginine residue in FliC, a pEtFliC construct in which the 91st asparagine was mutated to arginine (pEtFliC_N91R) was generated. Expression of the IL-1ß and NF-κB-p65 genes in the HINAE cells co-transfected with pEtFliC_N91R and pPoTLR5S was significantly higher than that in cells co-transfected with pEtFliC and pPoTLR5S. The results suggested that the 91st arginine residue of bacterial flagellin is involved in inflammatory response through TLR5S in teleosts. Thus, EtFliC improved by site-directed mutagenesis could be an effective adjuvant against E. tarda infection in Japanese flounder.

Molecular cloning of polymeric immunoglobulin receptor-like (pIgRL) in flounder (Paralichthys olivaceus) and its expression in response to immunization with inactivated Vibrio anguillarum.

In the present work, the polymeric immunoglobulin receptor-like (pIgRL) from flounder (Paralichthys olivaceus) was firstly cloned and identified. The full length cDNA of flounder pIgRL was of 1393 bp including an open reading frame of 1053 bp, and the deduced pIgRL sequence encoded 350 amino acids, with a predicted molecular mass of 39 kDa. There were two immunoglobulin-like domains in flounder pIgRL. In healthy flounder, the transcriptional level of pIgRL was detected in different tissues by real-time PCR, showing the highest level in the skin and gills, and higher levels in the spleen and hindgut. After flounders were vaccinated with inactivated Vibrio anguillarum via intraperitoneal injection and immersion, the pIgRL mRNA level increased firstly and then declined in all tested tissues during 48 h, and the maximum expression levels in the gills, skin, spleen and hindgut in immersion group, or in the spleen, head kidney, skin and gills in injection group, were higher than in other tested tissues. In addition, recombinant protein of the extracellular region of flounder pIgRL was expressed in Escherichia coli BL21 (DE3), and rabbit anti-pIgRL polyclonal antibodies were prepared, which specifically reacted with the recombinant pIgRL, and a 39 kDa protein confirmed as natural pIgRL by liquid chromatography-mass spectrometry in skin mucus of flounder. Co-immunoprecipitation assay and western-blotting demonstrated that the pIgRL, together with IgM, could be immunoprecipitated by anti-pIgRL antibody in gut, skin and gill mucus of flounder, suggesting the existence of pIgRL-IgM complexes. These results indicated that the flounder pIgRL was probably involved in the mucosal IgM transportation and played important roles in mucosal immunity.

RNA-seq and microRNA-seq analysis of Japanese flounder (Paralichthys olivaceus) larvae treated by thyroid hormones.

Flatfish pigmentation is a complex process, affected by environmental factors including light, nutrients, and hormones. Of those, the thyroid hormone has been reported to increase the albinism rate of Japanese flounder (Paralichthys olivaceus). However, the underlying mechanism remains unclear. In the present study, triiodothyronine (T3), thyroxine, and thiourea were introduced into P. olivaceus larvae from 16 to 57 days after hatching (DAH). By comparison of albinism rate, T3 treatment and control larvae of 42 DAH were chosen for mRNA and miRNA high-throughput sequencing analyses. A total of 337 miRNAs were identified via miRNA-seq, and 12 miRNAs exhibited significantly differential expression patterns in D42_T3 versus D42_Con (TPM > 10, fold change ≥ 1.5 or ≤ 0.67 and q ≤ 0.05). These differentially expressed miRNAs targeted 3658 putative genes, which further enriched to 10 GO terms (q < 0.05). RNA-seq identified 146 differentially expressed genes (DEGs) in D42_T3 versus D42_Con (|log2 fold change| > 1 and q < 0.005), including pigmentation-related genes such as the receptor tyrosine-protein kinase erbB-3, pro-opiomelanocortin A, and melanotransferrin, and the growth-related gene somatotropin. These DEGs were significantly enriched to 15 GO terms and 8 KEGG pathways (q < 0.05), which included several sugar metabolic pathways (glycolysis/gluconeogenesis and the pentose phosphate pathway). Integrated analysis revealed that 26 overlapping genes between DEGs and mRNAs were targeted by miRNAs. Furthermore, seven mRNA-miRNA pairs exhibited reversed regulation patterns. This provides important clues to understand the role of thyroid hormones in flatfish pigmentation.

Olive flounder CD276 (B7-H3) a coinhibitory molecule for T cells: Responses during viral hemorrhagic septicemia virus (VHSV) stimulation.

Coinhibitory pathways in the B7-CD28 family provide critical inhibitory signals that regulate immune homeostasis, defense and protect tissue integrity. CD276 (B7-H3) is an important immune checkpoint member of this family, which is induced on antigen-presenting cells (APCs), and plays an important role in the inhibition of T-cell function. We have characterized the CD276 gene of olive flounder, Paralichthys olivaceus. OfCD276 has an ORF of 912 bp that codes for 303 amino acids with a predicted molecular mass of 33 kDa. It is a type I transmembrane protein with a single extracellular V- and C-like Ig domains, a transmembrane region, and a highly diverse cytoplasmic tail. This gene was distinctly expressed in gill, spleen, and skin, and sparsely expressed in other tissues. Pathogen stimulation by VHSV revealed that transcription of OfCD276 was induced on early hours in liver and expressed late in head kidney, spleen, intestine and gill tissues. Flow cytometry analysis of leukocytes revealed the percentage of granulocytes and lymphocytes that expressed OfCD276 molecules on their cell surface was 85.1% and 3.1%, respectively. Our study shows a significant role played by this coinhibitory molecule that participate in the regulation of the cell mediated immune response.

Recombinant phosphoglucomutase and CAMP factor as potential subunit vaccine antigens induced high protection against Streptococcus iniae infection in flounder (Paralichthys olivaceus).

AIMS: The aim of this study was to screen vaccine candidates from virulence factors of Streptococcus iniae in flounder model. METHODS AND RESULTS: The immunogenicity of recombinant phosphoglucomutase (rPGM) and rCAMP factor was confirmed by Western blot. The percentage of surface membrane immunoglobulin-positive (sIg+ ) lymphocytes in peripheral blood leucocytes, the specific and total serum IgM and the activity of acid phosphatase (ACP) and peroxidase (POD) in flounder were determined with flow cytometry, ELISA and commercial enzyme activity kits, respectively, after intraperitoneal immunization with rPGM and rCAMP factor. The results showed that rPGM and rCAMP factor could induce significant rise in sIg+ lymphocytes, specific serum IgM and activities of ACP and POD. Additionally, the relative percent survival rate of the vaccinated flounder was 64 and 54% in challenge experiment using S. iniae, respectively. These results indicated that rPGM and rCAMP factor could evoke humoural and innate immune response in flounder and provide high-efficiency immunoprotection against S. iniae infection. CONCLUSIONS: Phosphoglucomutase (PGM) and CAMP factor were promising vaccine candidates against S. iniae in flounder. SIGNIFICANCE AND IMPACT OF THE STUDY: Phosphoglucomutase and CAMP factor have the potential to be vaccine candidates, which provide important information for us to develop the effective subunit vaccines, especially the multivaccine, against S .iniae in aquaculture.

Comparative performance of growth, vertebral structure and muscle composition in diploid and triploid Paralichthys olivaceus.

Growth, skeletal structure and muscle composition of cold-shock-induced triploid olive flounder Paralichthys olivaceus were investigated. The average values of total length and total weight of triploids were higher than those of diploids from 5 to 11 months posthatch (mph). The growth difference disappeared after 11 mph. The skeletal structure of flounder at 11 mph was observed by X-ray imaging method. There are four kinds of vertebral deformity including vertebrae fusion, one-sided compression, two-sided compression and vertically shifted. The trunk region (V8-18) and tailing end of the vertebral column were the predominant locations of deformity. In general, the frequencies of vertebral deformities in triploids (60.0%) were higher than those in diploids (33.3%, p < 0.05). Both the number of fish with deformed vertebrae and the average frequencies of deformed vertebrae in triploids were significantly higher than those in diploids (p < 0.05). The muscle tissues of diploid and triploid flounder at 11 mph contain the same types of fatty acid and free amino acid profiles. The number of fatty acids with significant higher contents in diploids and triploids was one and ten, respectively (p < 0.05). The contents of free amino acids showed no difference between triploid and diploid fish.

Development and Characterization of Monoclonal Antibodies to the 32 kDa Viral Attachment Protein of Lymphocystis Disease Virus and Their Neutralizing Ability in Vitro.

In previous research, a 32 kDa protein in lymphocystis disease virus (LCDV) was identified as viral attachment protein (VAP) that specifically interacted with the 27.8 kDa cellular receptor from flounder Paralichthys olivaceus gill (FG) cells, and the recombinant VAP (rVAP) was expressed in Escherichia coli strain BL21 (DE3). In this study, monoclonal antibodies (MAbs) against 32 kDa VAP are produced by immunization of BALB/c mice with the rVAP. Seven hybridoma secreting MAbs were screened by enzyme-linked immunosorbent assay, five of which designated as 1C6, 1C8, 3B5, 3D11 and 3H10 are cloned by the limiting dilution method, depending on the strongly positive results of ELISA. Western blotting analysis shows that the five MAbs can specifically react with the 32 kDa protein of LCDV and the purified 50 kDa rVAP, and the subtype of the MAbs is identified as IgG. Immunofluorescence results demonstrate that the specific fluorescence signals for LCDV appear in the cytoplasm of FG cells at 24 h post LCDV infection. Neutralization assay results indicate that pre-incubations of LCDV with the five MAbs can significantly decrease the LCDV copy numbers and delay the development of the cytopathic effect in FG cells, revealing that the five MAbs can neutralize the LCDV particles and block viral infection in vitro. The neutralizing MAbs against 32 kDa VAP would be useful for the study on the LCDV⁻host interaction and might be promising inhibitors of LCDV infection in fish.

The influence of concentration of inactivated Edwardsiella tarda bacterin and immersion time on antigen uptake and expression of immune-related genes in Japanese flounder (Paralichthys olivaceus).

Our previous work has demonstrated that the immune response of Japanese flounder was associated with the concentration of formalin-inactivated Edwardsiella tarda and immersion time. In order to further investigate the influence of immersion vaccine dose and bath time on the antigen uptake, formalin-killed Edwardsiella tarda bacterin was prepared and adjusted to four concentrations (109, 108, 107, 106 cfu ml-1) for 30, 60 and 90 min immersion in Japanese flounder model, respectively. Absolute quantitative real-time PCR was employed to examine the bacterin uptake in gill, skin, spleen and kidney at 3 and 6 h post vaccination. The results showed that the antigen uptaken in gills and skin were significant higher than spleen and kidney, and the antigen amounts in gill and skin both declined from 3 to 6 h, whereas the antigen amounts in spleen and kidney gradually increased. Significant higher antigen amounts were detected in 109-30, 109-60, 108-60, 108-90 and 108-90 groups than other groups (P < 0.05), especially the 108-60min group displayed the highest antigen uptaken. Meanwhile, the expression profiles of antigen recognization and presentation genes (MHCⅡα, TcRα, CD4-1), immunoglobulins (IgM, IgT), inflammatory cytokines (IL-1ß, IL-6), heat shock protein 70 (HSP70) and c-type lysozyme were analyzed using real-time PCR. On the whole, the transcription levels of the eight genes exhibited to be higher in 107-90, 108 and 109 cfu ml-1 groups than other groups (P < 0.05), especially the 108-60 group displayed the highest up-regulation. These results demonstrated that immersion with formalin-inactivated E. tarda, especially under 108-60 min condition could efficiently enhance the antigen uptake and the expression of immune-related genes, which provided evidences for an enhanced vaccination effects under an optimized combination of vaccine dose and immersion time.

Protective effects of CpG-ODN 2007 administration against Edwardsiella tarda infection in olive flounder (Paralichthys olivaceus).

In this study, we investigated the immunostimulatory and protective effects of CpG motif oligonucleotides (CpG-ODNs) against Edwardsiella tarda infection in olive flounder (Paralichthys olivaceus). Groups of fish injected with CpG-ODNs (1585, 1668, and 2007) or PBS (control) showed varying mortality rates in response to challenge with E. tarda. The survival rates of fish treated with CpG-ODN 1668 and 2007, which belonged to the same class type B, were 45% and 60%, respectively, with CpG-ODN 2007 showing the highest survival rate. Further analysis showed that the respiratory burst and bactericidal activities induced by CpG-ODN 2007 were higher than those in the control group (induced by non-CpG-ODNs) or in the group of fish induced by CpG-ODN 1585, which belonged to class type A. Additionally, the respiratory burst activity induced by CpG-ODN 2007 was higher than that induced by CpG-ODN 1668, despite similar bactericidal activity titers. In vivo experiments showed that CpG-ODN 2007 stimulation resulted in higher survival rates than CpG-ODN 1668 stimulation, possibly owing to differences in respiratory burst activity. In summary, we demonstrated that differences in CpG-motif or class type altered respiratory burst and bactericidal activities, resulting in differences in survival rates against E. tarda challenge in the olive flounder. Therefore, it is necessary to use CpG-ODNs optimized against E. tarda infection in olive flounder, because different CpG motifs belonging to the same class type have different effects.