Olive Flounder Paralichthys Olivaceus Research Articles

Hepcidin-1 in olive flounder (Paralichthys olivaceus): Gene expression, antimicrobial and therapeutic effects of synthetic peptides against bacterial and viral infections

Hepcidin is known to play an important role in iron metabolism and innate immunity as both a major regulator of iron homeostasis and, an antimicrobial peptide. In this study, we attempted to characterize the transcriptional and antimicrobial properties of hepcidin-1 in olive flounder (OF-hepcidin-1). Briefly, the gene expression level of OF-hepcidin-1 in olive flounder infected with viral hemorrhage septicemia virus (VHSV), Streptococcus parauberis, and Edwardsiella piscicida was quantified using real-time polymerase chain reaction (PCR) analysis. The antimicrobial activity and therapeutic effects of synthetic OF-hepcidin-1 were tested against several fish pathogens. The results showed that the expression of OF-hepcidin-1 mRNA was dramatically elevated in the liver, spleen, head kidney, and blood of infected olive flounder from 6 h post-challenge (hpc) to 7 days post-challenge (dpc), thus indicating its important role in innate immunity. Of note, a 133-fold increase in OF-hepcidin-1 mRNA was found in the blood of fish infected with VHSV at 6 hpc, while the VHSV concentration in the blood was near the border of detection limit; this indicates that OF-hepcidin-1 mRNA could be potentially used as an early infection bioindicator. OF-hepcidin-1 gene expression was significantly correlated with OF-IL-6 expression (p < 0.05), indicating that IL-6 might be closely involved in hepcidin expression in fish. Synthetic OF-hepcidin-1 exhibited antimicrobial activity against various fish pathogens including VHSV at a minimum dose of 50 μM fish−1. Further, OF-hepcidin-1 treatment in olive flounder infected with S. parauberis and VHSV showed a significantly lower infection rate and mortality (relative survival rate of 22.2% and 36.4%, respectively) than the control group, which suggests its potential usefulness in disease control applications.

Single and Multi-Strain Probiotics Supplementation in Commercially Prominent Finfish Aquaculture: Review of the Current Knowledge.

The Nile tilapia Oreochromis niloticus, Atlantic salmon Salmo salar, rainbow trout Oncorhynchus mykiss, olive flounder Paralichthys olivaceus, common carp Cyprinus carpio, grass carp Ctenopharyngodon idella and rohu carp Labeo rohita are farmed commercially worldwide. Production of these important finfishes is rapidly expanding, and intensive culture practices can lead to stress in fish, often reducing resistance to infectious diseases. Antibiotics and other drugs are routinely used for the treatment of diseases and sometimes applied preventatively to combat microbial pathogens. This strategy is responsible for the emergence and spread of antimicrobial resistance, mass killing of environmental/beneficial bacteria, and residual effects in humans. As an alternative, the administration of probiotics has gained acceptance for disease control in aquaculture. Probiotics have been found to improve growth, feed utilization, immunological status, disease resistance, and to promote transcriptomic profiles and internal microbial balance of host organisms. The present review discusses the effects of single and multi-strain probiotics on growth, immunity, heamato-biochemical parameters, and disease resistance of the above-mentioned finfishes. The application and outcome of probiotics in the field or open pond system, gaps in existing knowledge, and issues worthy of further research are also highlighted.

Saponin and chitosan-based oral vaccine against viral haemorrhagic septicaemia virus (VHSV) provides protective immunity in olive flounder (Paralichthys olivaceus)

Production losses of olive flounder (Paralichthys olivaceus) have increased owing to viral haemorrhagic septicaemia virus (VHSV) infection. In this study, we determined safe concentrations of orally administered saponin and chitosan by analysing serum enzyme (AST/ALT) levels as biochemical markers of hepatic injury. Furthermore, we demonstrated the efficacy, duration of protection, and safety of saponin and chitosan-based vaccines with inactivated VHSV (IV). Oral administration of saponin, chitosan, and their combination did not induce fish mortality at all tested concentrations (0.29, 1.45, and 2.9 mg/g of fish body weight/day) 10 days after administration. However, AST level was high at a dose >0.29 mg/g of fish body weight/day. Both saponin and chitosan were found to be safe and acceptable for vaccination studies at a dose of 0.29 mg/g of fish body weight/day. Administration of IV alone did not induce protection at 2 and 4 weeks post vaccination (wpv). Olive flounders administered saponin + IV and chitosan + IV vaccines had higher immunity against VHSV with relative percentage survival (RPS) of 12.5–7.5% and 0–20.1%, respectively; however, additional immunisation with combination of saponin + chitosan + IV clearly enhanced the protection with RPS values of 10–15%, 26.7%, 42.9%, and 37.5% at 4, 8, 12, and 20 wpv, respectively. Although the RPS value of oral immunisation was not comparable to that of injectable vaccines, the manufacturing process is simple and oral administration causes less stress to juvenile fish. To investigate the development of a protective immune response, olive flounder were re-challenged with VHSV (107.8 TCID50/fish) at 70 days postinfection; 100% of the previously unexposed fish died, whereas 80–100% of the previously immunised fish survived. Our results showed the possibility of developing preventive measures against VHSV using saponin and chitosan-based oral vaccines with inactivated virus.

Downregulated pol-miR-140-3p induces the expression of the kinesin family member 5A against Streptococcus parauberis infection in olive flounder

MicroRNAs (miRNAs) are small non-coding RNAs that participate in various biological and cellular processes by regulating target gene expression. miRNAs are also known to play vital roles in the pathogenesis of various diseases, including infections, as well as the disease progression and defense responses. In this study, we examined the expression levels of pol-miR-140-3p and its target gene, kinesin family member 5A (KIF5A), in association with the Streptococcus parauberis (S. parauberis) infection, a major bacterial pathogen that causes streptococcosis in olive flounder (Paralichthys olivaceus). KIF5A is a heavy chain isoform of kinesin-1, which is known to be brain-specific, and this study is the first examination of KIF5A expression related to the regulation of miRNA in olive flounder (named PoKIF5A). There were significant differences in expression levels between infected and healthy olive flounder as the expression of pol-miR-140-3p in the infected fish was lower than that in the control, while the expression of PoKIF5A was higher in the infected fish than in the healthy controls. These contradictory results suggest that downregulated pol-miR-140-3p induces the expression of PoKIF5A against S. parauberis infection in olive flounder.

Evaluating the efficacy of partially substituting fish meal with unfermented tuna by-product meal in diets on the growth, feed utilization, chemical composition and non-specific immune responses of olive flounder (Paralichthys olivaceus)

Fish meal (FM) usage in aquafeed is fraught with cost and environmental challenges. Alternatives to FM must be readily available and with adequate protein. This 8-weeks study explored the utilization of tuna by-product meal (TBM) without fermentation as a substitute for FM in diet on growth, feed utilization, chemical composition, plasma chemistry, and non-immune responses of olive flounder (Paralichthys olivaceus). Four hundred juveniles (initial weight of 14.7 g) were randomly distributed to 20 tanks (20 fish per tank). The control (Con) diet contained 65% FM without replacement, and in the TBM10, TBM20, TBM30, TBM40, and TBM50 diets, FM was substituted with 10%, 20%, 30%, 40%, and 50% TBM, respectively. Each diet was fed to triplicate groups of fish, but quadruplicate groups for the Con and TBM30 diets. Survival was not different among the experimental diets. Superior weight gain, specific growth rate (SGR), feed efficiency ratio (FER) and protein efficiency ratio were observed in fish fed the TBM40 and TBM50 diets to fish fed all other diets. Condition factor of fish fed the TBM40 and TBM50 diets was higher than that of fish fed the Con diet. Feed consumption, chemical composition, and amino acid profiles of the whole body fish, except for the tryptophan content, viscerosomatic and hepatosomatic indices, plasma biochemical parameters, non-specific immune responses were not affected by the experimental diets. In conclusion, FM up to 50% can be replaced with TBM without impairing growth, feed utilization, nutritional composition, plasma chemistry, and non-specific immune responses of olive flounder when 65% FM was included. The greatest weight gain, SGR, and FER of fish were estimated to be 47.1%, 48.6%, and 46.3% of dietary substitution of FM with TBM, respectively.

Genome-wide association study (GWAS) of growth traits in olive flounder (Paralichthys olivaceus)

Traits linked to growth performance in farmed fish are major drivers of profitability in aquaculture systems. Given the importance of growth-related traits, there have been efforts to unravel the genomic architecture of growth traits and to identify key genes or genomic regions useful as selection markers in various species. Such markers would increase the accuracy of selection and improve rates of genetic progress for growth improvement in breeding programs. Olive flounder (Paralichthys olivaceus) has a considerable market value in Asia and is mainly cultured in South Korea. To facilitate a breeding program for this species, a genome-wide association study (GWAS) was conducted to identify single nucleotide polymorphisms (SNP) associated with growth traits. In total, 1800 fish were measured for body weight (BW), total length (TL), and maximum body depth (BD). Fish were genotyped using the Affymetrix® Axiom® myDesign™ Genotyping Arrays; 1009 fish and 58,756 SNPs remained after the final quality control check. According to the phenotypes, female fish recorded higher BW, TL, and BD than male counterparts of the same age, revealing a sex-dependent growth pattern. There was a strong positive phenotypic correlation (0.945–0.965) and a moderate genotypic correlation (0.286–0.332) between the three growth performance traits of the 1009 fish. Eight SNPs were identified above the significant (p < 8.5 × 10−7) and suggestive (p < 1 × 10−5) thresholds for all traits, and they were mapped primarily onto chromosomes 23 and 24. According to the GWAS analysis, genotypes at loci AX-419206420 and AX-419307742 significantly affected the growth performance of the population. Gene ontology (GO) analysis was performed on 30 candidate genes with notable growth performance effects (p < 1 × 10−4) and GO terms related to neuron development, molecule binding and cytoskeleton organization were highly enriched. Our findings may contribute to understanding of the molecular basis of growth regulation and promote the use of SNP-based genomic selection in flounder culture.

Effects of Bacillus SW1-1 coated diets on innate immunity and disease resistance of olive flounder Paralichthys olivaceus against Edwardsiella tarda infection

Bacillus SW1-1 is a probiotic isolated from shrimp intestines. We investigated the effects of Bacillus SW1-1 coated diets on the growth, feed utilization, innate immunity, hematological parameters and resistance to Edwardsiella tarda in olive flounder (Paralichthys olivaceus). A commercial diet was used as the control (AP0) and two other diets were prepared by coating 0.25% (AP25) or 0.50% (AP50) probiotic powder which contains 1.0 × 107 CFU/g Bacillus SW1-1. Four replicate groups of olive flounder (153 ± 2 g) were fed one of the diets for 12 weeks. Growth performance and feed utilization of the fish were not significantly affected by the dietary Bacillus SW1-1. After the challenge with E. tarda, AP50 group showed significantly higher survival than AP0 and AP25 groups. Innate immunity and anti-oxidant capacity of the fish were not significantly affected after the feeding trial. However, after the E. tarda challenge, the innate immune parameters (immunoglobulin, lysozyme and anti-protease) were significantly improved in fish fed AP25 and AP50 diets compared to those in fish fed AP0 diet. After the challenge test, significantly lower glu-cose level was observed in AP50 group compared to AP0 group. These results indicate that dietary supplementation of Bacillus SW1-1 could increase the disease resistance of olive flounder against E. tarda infection. The optimum coating levels of Bacillus SW1-1 needs to be further elucidated.

Taurine alleviates endoplasmic reticulum stress, inflammatory cytokine expression and mitochondrial oxidative stress induced by high glucose in the muscle cells of olive flounder (Paralichthysolivaceus)

The aim of the present study was to evaluate the effects of taurine on endoplasmic reticulum stress, inflammatory cytokine expression and mitochondrial oxidative stress induced by high glucose in primary cultured muscle cells of olive flounder (Paralichthys olivaceus). Three experimental groups were designed as follows: muscle cells of olive flounder incubated with three kinds of medium containing 5 mM glucose (control), 33 mM glucose (HG) or 33 mM glucose + 10 mM taurine (HG + T), respectively. Results showed that taurine addition significantly alleviated the decreased activity of superoxide dismutase (SOD) and the ratio of reduced to oxidized glutathione (GSH/GSSG) induced by high glucose. The increase of cellular reactive oxygen species (ROS), malondialdehyde content and cell apoptosis induced by high glucose were alleviated by taurine. Besides, gene expression of glucose-regulated protein 78, PKR-like ER kinase, tumor necrosis factor-α, interleukin-6, interleukin-1β, interleukin-8, muscle atrophy F-box protein and muscle RING-finger protein 1 were significantly up-regulated in the HG group, and taurine addition decreased the expression of these genes. High glucose led to the swelling of the endoplasmic reticulum (ER). Meanwhile, the nuclear translocation of nuclear factor κB (NF-κB) and the release of cytochrome C from mitochondria induced by high glucose were suppressed by taurine addition. These results demonstrated that taurine alleviated ERS, inflammation and mitochondrial oxidative stress induced by high glucose in olive flounder muscle cells. The ROS production, NF-κB signaling pathway and mitochondria function were the main targets of the biological effects of taurine under high glucose condition.

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.

Transcriptional Expression Profiles of Olive Flounder (Paralichthys olivaceus) Toll-like Receptor 3 (TLR3) and TLR22 in Response to Extracellular Poly I:C Stimuli

Toll-like receptors (TLRs) recognize extracellular and intracellular double-stranded RNA (dsRNA) and play crucial roles in innate and adaptive immune systems. Here, we aimed to investigate the changes in the transcriptional profile of TLR3 and TLR22 genes of olive flounder (Paralichthys olivaceus) in response to polyinosinic:polycytidylic acid (poly I:C; a dsRNA analog) stimuli in vivo, using healthy olive flounders and in vitro, using Hirame natural embryonic (HINAE) cells. The role of JfTLR3 and JfTLR22 in inducing type I interferon response in HINAE cells was analyzed through construction and transfection of short-hairpin RNA (shRNA)-producing vector targeting the JfTLR22 and JfTLR3. Poly I:C stimuli upregulated the JfTLR3 and JfTLR22 genes both in vivo and in vitro, and JfTLR22 showed an earlier (at 6 and 12 h post-stimulation) response than JfTLR3, suggesting that JfTLR22 is a more sensitive receptor and recognizes the extracellular dsRNA more quickly than JfTLR3. After 12 h of poly I:C stimulation, the expression of ISG15 and Mx genes in HINAE cells harboring TLR22-targeting shRNA vector was markedly reduced, wherein the reduction in expression of these genes was weakly reduced in HINAE cells carrying TLR3-targeting shRNA vector. These results suggested that JfTLR22 plays an important role, wherein JfTLR3 partially induces type I interferon response to extracellular dsRNA at 12 h after poly I:C stimulation.

Comparative toxicity study of waterborne two booster biocides (CuPT and ZnPT) on embryonic flounder (Paralichthys olivaceus)

A new generation of booster biocides that include metal pyrithiones (PTs) such as copper pyrithione (CuPT) and zinc pyrithione (ZnPT) are being used as tributyltin alternatives. In the marine environment, ZnPT can easily transchelate Cu to form CuPT, and the environmental fate and persistence of these two metal pyrithiones are closely related. Although some data on the toxicity of biocides on marine fish are available, little is known about their toxicity and toxic pathway. We thus compared the toxic effects of CuPT and ZnPT on embryonic olive flounder (Paralichthys olivaceus) by investigating their adverse effects based on developmental morphogenesis and transcriptional variation. In our study, the toxic potency of CuPT was greater with respect to developmental malformation and mortality than ZnPT. Consistent with the developmental effects, the expression of genes related to tail fin malformation (including plod2, furin, and wnt3a) was higher in embryonic flounder exposed to CuPT than in those exposed to ZnPT. Genes related to muscle and nervous system development exhibited significant changes on differential gene expression profiles using RNA sequencing (cutoff value P < 0.05). Gene ontology analysis of embryos exposed to CuPT revealed affected cellular respiration and kidney development, whereas genes associated with cell development, nervous system development and heart development showed significant variation in embryonic flounder exposed to ZnPT. Overall, our study clarifies the common and unique developmental toxic effects of CuPT and ZnPT through transcriptomic analyses in embryonic flounder.

Genetic diversity and signatures of selection in the mito-gynogenetic olive flounder Paralichthys olivaceus revealed by genome-wide SNP markers

The artificial gynogenesis technique has been proven to be an effective genetic tool and has been widely used in fish breeding. The evaluation of the genetic basis of the artificial gynogenetic population is essential for breeding and farming, and the detection of the signatures of selection is potential to elucidate genes and mutations associated with important phenotypic traits. Olive flounder Paralichthys olivaceus is a commercially important flatfish species cultured in China, Japan, and Korea, and artificial gynogenesis has been used for its genetic breeding. In this study, the genetic variation of the artificial mito-gynogenetic population and common population of the flounder were analyzed using SNP markers with 2b-RAD genotyping technique. A total of 162 mito-gynogenetic and 90 common flounders were used, and 39,018 high-quality genome-wide single nucleotide polymorphisms (SNPs) were identified across two populations. The average observed heterozygosity (Ho) and average expected heterozygosity (He) of SNP were 0.1895 and 0.2413, and 0.0222 and 0.2974 in the common and mito-gynogenetic populations, respectively. Most loci significantly deviated from Hardy-Weinberg equilibrium (HWE) after Bonferroni correction in the former population. However, the genetic distance between the two populations suggested no significant genetic differentiation. Furthermore, analysis of run of homozygosity (ROH) showed low level of genomic inbreeding in the common population (inbreed coefficient was 0.2171) compared to higher inbreeding in the mito-gynogenetic population (inbreed coefficient was 0.9227). Short ROHs were more frequent in the common population, while long ROHs were more frequent in the mito-gynogenetic population, providing insight in the developmental history of the flounder. A total of 580 genes with putative selection signatures were identified across the two populations based on selective sweeps. These genes were involved in 39 KEGG pathways associated with some important traits, such as reproduction (e.g., sox8 and sox9) and immune response (e.g., dctn5 and socs-1). These results would be helpful for molecular marker-assisted breeding and conservation of genetic resources in the olive flounder and other marine culture fish.

Animal and plant proteins as alternative ingredients in diets for sub‐adult olive flounder Paralichthys olivaceus at farm conditions

A 20-week feeding trial was performed to evaluate the optimum level of animal and plant protein sources as substitutes of fishmeal (FM) in the diets of sub-adult olive flounder (Paralichthys olivaceus) under farmed conditions. Groups of ninety fish averaging 478 ± 4.7 g (mean ± SD) were randomly distributed into four treatments in triplicates. An FM-based diet was used as control (CON) and three other diets replaced 20% (APP20), 30% (APP30) and 40% (APP40) of FM with animal (tankage meal and poultry by-product meal) and plant (soy protein concentrate) protein sources. Weight gain, specific growth rate and protein efficiency ratio were not significantly different among fish fed all the experimental diets (p > 0.05). The feed efficiency of fish fed the APP40 diet was significantly lower than those of fish fed the other experimental diets (p < 0.05). Non-specific immune responses such as superoxide dismutase activity and lysozyme activity of fish fed all the experimental diets were not significantly different (p > 0.05). Intestinal villi length of fish fed the CON diet was significantly higher than those of fish fed the other diets (p < 0.05). Based on the results of growth performances and the non-specific immune responses, the dietary combination of animal and plant protein sources could replace up to 40% of FM in sub-adult olive flounder.

Toxic effects of waterborne cadmium exposure on hematological parameters, oxidative stress, neurotoxicity, and heat shock protein 70 in juvenile olive flounder, Paralichthysolivaceus

Cadmium-induced toxicity can affect fish embryo development, ion homeostasis regulation, energy metabolism, maturation and growth, stress response, and immunity. However, studies on the toxic effects of cadmium exposure to aquatic animals, particularly olive flounder (Paralichthys olivaceus), are limited. In this study, juvenile P. olivaceus (mean length, 12.9 ± 1.3 cm; mean weight, 23.1 ± 3.2 g) was exposed to waterborne cadmium (0, 50, 100, 200, and 400 μg/L) for 10 d. Hematological parameters, including hematocrit value and hemoglobin level, in P. olivaceus were significantly decreased after waterborne cadmium exposure. Plasma components such as calcium, glucose, cholesterol, glutamic-oxaloacetic transaminase, and glutamic-pyruvic transaminase were significantly altered via cadmium exposure. The activities of antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione S-transferase, were significantly altered in P. olivaceus after cadmium exposure. Acetylcholinesterase activity was significantly inhibited upon waterborne cadmium exposure. Hepatic heat shock protein 70 was significantly upregulated in P. olivaceus after waterborne cadmium exposure. Therefore, waterborne cadmium at concentrations of >100 or 200 μg/L can induce physiological toxicity in P. olivaceus via changes in hematological parameters, antioxidant enzymes, neurotransmitters, and stress indicators.

CRISPR/Cas9-mediated generation of auxotrophic Edwardsiella piscicida mutants and immunization in olive flounder (Paralichthys olivaceus)

Edwardsiella piscicida has been a cause of mass mortality in cultured fish. In this study, to produce auxotrophic E. piscicida mutants, a CRISPR/Cas9 system was used instead of the traditional sacB-based allelic exchange method. Under the optimal CRISPR engineering condition, we could efficiently produce either alr or asd gene knockout E. piscicida auxotrophic mutants, and this genome editing process was much simpler and faster than the allelic exchange method. The simultaneous knockout of double auxotrophic genes (alr and asd) and the insertion of a foreign gene expression cassette in E. piscicida chromosome were also successfully performed using the established CRISPR/Cas9 system. Furthermore, to enhance the possibility to get permission as a commercial vaccine, we produced an auxotrophic E. piscicida mutant having only one nucleotide-deleted alr gene (E. piscicida △alr-1). Olive flounder (Paralichthys olivaceus) fingerlings immunized with 1 × 106 and 1 × 105 CFU/fish of E. piscicida △alr-1 showed the superior ability in the induction of serum agglutination activity and in the protection against E. piscicida compared to killed E. piscicida. However, olive flounder immunized with 1 × 107 CFU/fish of E. piscicida △alr-1 showed high mortality far before the challenge, and the isolated E. piscicida from moribund and dead fish had the wild type alr gene, suggesting the reversion of one base-deleted alr gene to original form by a second mutation in olive flounder. Therefore, investigation on the minimum number of edited nucleotide for stable maintenance of E. piscicida mutants should be further conducted.

Evaluation of Dietary Probiotic Bacteria and Processed Yeast (GroPro-Aqua) as the Alternative of Antibiotics in Juvenile Olive Flounder Paralichthys olivaceus.

We investigated the three probiotic bacteria and a processed yeast (GroPro-Aqua) as the replacers of antibiotics in juvenile olive flounder. A total of seven diets were used, that is, one basal or control (CON) diet; and six other diets, of which, three diets were prepared by supplementing probiotic bacteria such as Bacillus subtilis WB60 (BSWB60) at 1 × 108 CFU/g diet, Bacillus subtilis SJ10 (BSSJ10) at 1 × 108 CFU/g diet, and Enterococcus faecium SH30 (EFSH30) at 1 × 107 CFU/g diet; one diet with processed yeast (GRO) at 0.35% diet; and two other diets were supplemented with oxytetracycline (OTC) and amoxicillin (AMO) at 4 g/kg of each. Triplicate groups of fish (average 12.1 g) were fed one of the diets for eight weeks. At the end of the feeding trial, the fish that were fed the probiotic bacteria-supplemented diets had a significantly higher final weight, weight gain, and specific growth rate compared to the CON, OTC, and AMO diets. Fish that were fed the GRO diet had significantly higher feed efficiency and protein efficiency ratios than those of the fish that were fed the CON diet. Serum glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, glucose, and total protein were not affected by the diets. Lysozyme activity in fish that were fed the BSSJ10, BSWB60, and EFSH30 diets were significantly higher compared to the CON and OTC diets, whereas myeloperoxidase activity of fish fed the BSWB60 and EFSH30 diets were significantly higher than those of fish fed the CON and AMO diets. Flounder growth hormone gene expressions of fish that were fed BSWB60 and GRO diets were significantly higher compared to the CON, OTC, and AMO diets. The interleukin-1β gene expression of fish that were fed the BSSJ10, BSWB60, EFSH30, OTC, and GRO diets was significantly higher than those of fish fed the CON diet. The interleukin-10 gene expression of fish that were fed the BSSJ10, EFSH30, and GRO diets was significantly higher than those of fish fed the CON and AMO diets. Posterior intestinal histology of fish showed significantly higher villi length in fish that were fed the BSSJ10, BSWB60, EFSH30, and GRO diets compared to the CON diet. After 15 days of challenge test with pathogenic bacteria Edwardsiella tarda, the cumulative survival rate of fish that were fed the BSSJ10, BSWB60, EFSH30, and GRO diets were significantly higher than those of fish that were fed the CON diet. Overall, the results indicate that dietary supplementation of B. subtilis (108 CFU/g diet), E. faecium (107 CFU/g diet), and processed yeast (GroPro-Aqua at 0.35% diet) could replace the antibiotics in terms of improving growth, immunity, gut health, and disease resistance in juvenile olive flounder.

Effect of low pH and salinity conditions on the antioxidant response and hepatocyte damage in juvenile olive flounder Paralichthys olivaceus

Climate change due to increasing CO2 emissions results in the increase in water temperatures, which is accompanied by the decrease in pH and salinity levels of the ocean. Ocean acidification reflects the gradual pH reduction due to changes in the carbon chemistry, which is caused by the increase in anthropogenic CO2 emissions. The subsequent changes in the water temperatures and carbon chemistry of the oceans affect the survival and distribution of aquatic animals. In this study, we analyzed the levels of cortisol, superoxide dismutase, catalase, and caspase-3 in the plasma of juvenile olive flounder Paralichthys olivaceus under combined hyposalinity and acidification. To evaluate the physiological response to these changes, the superoxide dismutase activity and apoptosis were analyzed in the liver cells. Hyposalinity caused oxidative stress and cell damage, while also activating the antioxidant system. Environmental acidification affected the stress response and antioxidant mechanism of P. olivaceus in the early stage of acclimation but did not appear to exceed hyposalinity stress. These findings suggest that a hyposaline environment may be a stronger environmental stressor than an acidifying environment for P. olivaceus, and will help understand the capacity of P. olivaceus to cope with expected future ocean acidification.

Establishment and Application of Four Long-Term Culture Cell Lines of the Olive Flounder Paralichthys Olivaceus Blastocysts

Establishment and Application of Four Long-Term Culture Cell Lines of the Olive Flounder Paralichthys Olivaceus Blastocysts

Physical Abnormalities of Olive Flounder (Paralichthys olivaceus) Juvenile Found During Sorting Process in Anmyeon, South Korea

Olive flounder (Paralichthys olivaceus) is the most popular consumption fish in South Korea. Korean consumes flounder as sashimi, spicy soup, stew and cutlet. Due to its popularity; Korean considers Olive flounder as the nation’s raw fish. Despite advanced aquaculture technique, modern facilities and massive production, fingerling size grading and abnormal sorting are conducted by traditional method. This study was conducted by observation. A group of 5 to 6 person worked meticulously to sort out abnormal fingerling and juvenile. Sorting is based on physical traits such as the shape and structure of head, jaw, operculum, fin, body; body coloration, pigmentation and length. Due to the exclusiveness and technicality of this profession, information and standard in grading and sorting has been lacking. This research provides the information about sorting criteria of Olive flounder juvenile and detail description of physical abnormalities being sorted out.

Potential Efficacy of Chitosan-Poly (Lactide-Co-Glycolide)-Encapsulated Trivalent Immersion Vaccine in Olive Flounder (Paralichthys olivaceus) Against Viral Hemorrhagic Septicemia Virus, Streptococcus parauberis Serotype I, and Miamiensis avidus (Scuticociliate).

Olive flounder (Paralichthys olivaceus) is the most valuable aquaculture species in Korea, corresponding to ~60% of its total production. However, infectious diseases often break out among farmed flounders, causing high mortality and substantial economic losses. Although some deleterious pathogens, such as Vibrio spp. and Streptococcus iniae, have been eradicated or contained over the years through vaccination and proper health management, the current disease status of Korean flounder shows that the viral hemorrhagic septicemia virus (VHSV), Streptococcus parauberis, and Miamiensis avidus are causing serious disease problem in recent years. Furthermore, these three pathogens have differing optimal temperature and can attack young fingerlings and mature fish throughout the year-round culture cycle. In this context, we developed a chitosan-poly(lactide-co-glycolide) (PLGA)-encapsulated trivalent vaccine containing formalin-killed VHSV, S. parauberis serotype-I, and M. avidus and administered it to olive flounder fingerlings by immersion route using a prime-boost strategy. At 35 days post-initial vaccination, three separate challenge experiments were conducted via intraperitoneal injection with the three targeted pathogens at their respective optimal temperature. The relative percentages of survival were 66.63%, 53.3%, and 66.75% in the group immunized against VHSV, S. parauberis serotype-I, and M. avidus, respectively, compared to the non-vaccinated challenge (NVC) control group. The immunized fish also demonstrated significantly (p < 0.05) higher specific antibody titers in serum and higher transcript levels of Ig genes in the mucosal and systemic tissues than those of NVC control fish. Furthermore, the study showed significant (p < 0.05) upregulation of various immune genes in the vaccinated fish, suggesting induction of strong protective immune response, ultimately leading to improved survival against the three pathogens. Thus, the formulated mucosal vaccine can be an effective prophylactic measure against VHS, streptococcosis, and scuticociliatosis diseases in olive flounder.