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Sustainable methods that increase farmed fish yield while controlling infections are required to prevent economic losses in aquaculture farms. In this study, we evaluated the effects of betaine-supplemented (0%, 0.1%, 0.5%, and 1.0%) feed on the growth and immunity of the olive flounder Paralichthys olivaceus. Feed conversion ratios, post-infection cumulative mortality rates and innate immune responses were monitored. Weight gain was significantly higher with 0.5% and 1.0% than with 0% and 0.1% betaine-supplemented feed. Lysozyme activity was highest with 1.0% betaine. Respiratory burst activity was highest with 0.5% and 1.0% betaine. Serum bactericidal activity against Edwardsiella tarda was highest with 1.0% betaine (40% increase in survival rates compared with those in the control). Furthermore, serum virucidal activity against the viral haemorrhagic septicaemia virus (VHSV) was higher with 1.0% betaine than with other concentrations. With 0.5% and 1.0% betaine, the survival rates against VHSV were higher than those in the control until day 11, after which they declined. Our study suggests that betaine is a promising agent for promoting the growth of and enhancing immunity against E. tarda in olive flounders. Our findings may further contribute to developing necessary alternatives to conventional antibiotics in fish farming.

Background Butaphosphan (BTP) has recently been introduced into the Korean aquaculture sector as a stress-attenuating agent. In this study, a sensitive chemical analytical method was established for the detection of BTP in the olive flounder (Paralichthys olivaceus) tissues. Methods Utilizing a method employing liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), detection sensitivity, specificity, and precision were satisfactorily established. Temporal changes in the BTP plasma and muscle concentrations were assessed after a single intramuscular injection of BTP (50 and 150 mg/kg) to the olive flounder maintained at 13 °C or 22 °C. Results High BTP plasma levels were achieved immediately after the injection, and the drug was rapidly eliminated. Additionally, plasma BTP levels were markedly dependent on the elimination rate, which, in turn, seemed dependent on the water temperature, with the drug elimination half-life and mean residence time significantly shorter at 22 °C than 13 °C. Overall, muscle BTP levels were markedly lower than the plasma levels. Notably, muscle levels were not influenced by water temperatures. Muscle BTP concentrations were used to estimate the necessary withdrawal period for drugs used in food fish, with BTP levels maintained far below the possible hazardous limit. Conclusions In conclusion, the established LC-MS/MS method can be used for BTP residue detection with high sensitivity and reproducibility.

Nitrite concentrations can reach high levels in indoor aquaculture systems, thus it is vital to determine the nitrite tolerance of aquaculture fish species. Here, juvenile hybrid groupers (Epinephelus lanceolatus ♂ × Epinephelus fuscoguttatus ♀, Family: Serranidae) were exposed to waterborne nitrite at 0, 10, 20, 40, and 80 mg NO2−/L for 2 weeks. Nitrite exposure caused significant reductions in hematocrit and hemoglobin levels, significant increases in plasma calcium and plasma ALP levels, but had no significant effects on magnesium and total protein levels. Of the antioxidant responses investigated, SOD activity increased significantly in the liver and gills, but GST activity and GSH levels were significantly inhibited by nitrite exposure. Stress indicators, such as plasma cortisol and HSP 70 levels, were significantly stimulated by nitrite exposure. In brief, nitrite exposure over 20 mg NO2−/L had toxic effects and affected the hematological properties, antioxidant responses, and stress indicators of juvenile hybrid groupers.

Butaphosphan {[1-(butylamino)-1-methylethyl]-phosphonic acid (BTP)}-cyanocobalamin [vitamin B12 (C)], a combination drug comprising an organic source of phosphorus and a synthetic form of vitamin B12, is used to attenuate stress responses in livestock. This study was performed to examine the effects of BTP-C in olive flounder Paralichthys olivaceus under normal and stressful conditions. P. olivaceus was intramuscularly injected with BTP-C (50–300 mg BTP-C/kg body weight) under normal conditions and changes in muscle adenosine nucleotides examined. BTP-C increased adenosine 5′-triphosphate (ATP) levels; this effect was more pronounced at 22 °C than at 13 °C. Serum transaminase levels were also elevated at a dose of 300 mg BTP-C/kg, which suggested possible hepatotoxicity of the drug. Effects of BTP-C on stress were examined by injecting it at a rate of 50 mg/kg. BTP-C partially prevented hypothermia- and hypoxia-induced reduction in feeding. Serum immunoglobulin M levels were increased by BTP-C in these stress tests. Although BTP-C did not affect hypothermia-induced cortisol elevation or lysozyme reduction, changes in these parameters due to hypoxia were alleviated by the drug. Stress-attenuating effects of BTP-C were also confirmed in fish farms following injection of an antibiotic. These results indicate that BTP-C may be useful against various forms of stress in fish. Accelerated ATP production following the administration of BTP-C is one possible mechanism resulting in the attenuation of stress effects. The clinically effective dose of BTP-C, 50 mg/kg, did not cause any marked toxicity in P. olivaceus.

Lead (Pb) is a highly toxic metal in aquatic environments. Fish are at the top of the food chain in most aquatic environments, and are the most susceptible to the toxic effects of Pb exposure. In addition, fish are one of the most abundant vertebrates, and they can directly affect humans through food intake; therefore, fish can be used to assess the extent of environmental pollution in an aquatic environment. Pb-induced toxicity in fish exposed to toxicants is primarily induced by bioaccumulation in specific tissues, and the accumulation mechanisms vary depending on water habitat (freshwater or seawater) and pathway (waterborne or dietary exposure). Pb accumulation in fish tissues causes oxidative stress due to excessive ROS production. Oxidative stress by Pb exposure induces synaptic damage and neurotransmitter malfunction in fish as neurotoxicity. Moreover, Pb exposure influences immune responses in fish as an immune-toxicant. Therefore, the purpose of this review was to examine the various toxic effects of Pb exposure, including bioaccumulation, oxidative stress, neurotoxicity, and immune responses, and to identify indicators to evaluate the extent of Pb toxicity by based on the level of Pb exposure.

Juvenile Paralichthys olivaceus (mean length 7.29 ± 0.59 cm, mean weight 2.41 ± 0.35 g) were exposed to several concentrations of ammonia (0, 6.25, 12.5, 25, 50, and 100 mg/L), nitrite (0, 50, 100, 200, 400, and 800 mg/L), and nitrate (0, 250, 500, 1000, 2000, and 4000 mg/L) for 96 h in 20-L glass tanks. Lethal concentration 50% (LC50) was determined after removing and counting dead fish at 0, 3, 6, 12, 24, 48, 72, and 96 h of exposure. Exposure was significantly toxic to P. olivaceus, and LC50 at 96 h was 26.008 mg/L for ammonia, 768.078 mg/L for nitrite, and 1431.343 mg/L for nitrate. The toxicity profile found for P. olivaceus juveniles was ammonia > nitrite > nitrate. For antioxidant activity analysis such as superoxide dismutase (SOD) and catalase (CAT) activity, liver and kidney tissues were dissected after 96 h of exposure. In liver and kidney tissues, SOD activity was significantly increased at 25 mg/L of ammonia, above 400 mg/L of nitrite, and at 1000 mg/L of nitrate. At these concentrations, CAT activity also increased, except in the kidney, where no change in CAT activity was detected under exposure to nitrate. The results of this study suggest that exposure to nitrogenous compounds such as ammonia, nitrite, and nitrate can induce significant toxicity and alterations in the antioxidant responses of P. olivaceus.

We examined the effect of acute air exposure on the hematological characteristics and physiological stress responses of the olive flounder (Paralichthys olivaceus) and Japanese croaker (Nibea japonica). Fish were exposed to air for 30 s (30S), 90 s (90S), and 180 s (180S). The control group was not exposed to air after capture. Experiments were performed in triplicate. The water exchange rate was 32 times the water volume of the tank and dissolved oxygen was maintained at over 5 mg/L. After air exposure (AE), fish were kept in the experimental tanks for 24 h. Blood samples were taken from the fish at 0 h (before air exposure) and at 1, 6, and 24 h AE. We found that the olive flounders and the Japanese croakers differed in their primary (hormonal) and secondary (hydromineral and metabolic) responses to the air exposure. Cortisol levels were lower in the olive flounders than in the Japanese croakers. In the 180S group, the olive flounder cortisol levels were increased by 62-fold whereas the Japanese croaker cortisol levels were increased by 75-fold. The cortisol levels increases were associated with elevated glucose levels in both olive flounders and Japanese croakers. Olive flounders and Japanese croakers (except in 180S) had increased glucose levels that recovered to a stable level or showed a decreasing trend until 24 h AE. Several studies have reported on the resilience of flounders to short-term stress. This was confirmed in the present study by the fast recovery of elevated cortisol, glucose, and lactic acid levels that were induced by the stressor. Compared to the stress response in the olive flounder, the Japanese croaker exhibited a stronger response associated with the cortisol, glucose, and lactic acid levels

Background Live fish import may lead to the unintended introduction of pathogens. We examined the monthly distribution of microbial pathogens in ornamental finfish imported into South Korea over a 6-month period. Results Vibrio alginolyticus was detected in one lemon damsel in June and July; V. vulnificus was detected in one lemon damsel, one caerulean damsel, and one pearl-spot chromis and one ocellaris clownfish in July, April, and May, respectively; Photobacterium damselae was detected in one ocellaris clownfish and one caerulean damsel in June and July, respectively; V. anguillarum was detected in one pearl-spot chromis in February; V. harveyi was detected in one ocellaris clownfish and two mandarin fish in February and April, respectively; Yersinia ruckeri was detected in a pearlscale goldfish group in June and July and in two colored carp groups in July; and Lactococcus garvieae was detected in a lemon damsel group and a sutchi catfish group in July and May, respectively. European catfish virus, the only viral pathogen detected, was found in two sutchi catfish groups in May. Conclusion This study is the first to identify pathogenic species and the presence or absence of pathogens (non-quarantine diseases) in imported ornamental finfish. These results demonstrate that various pathogens with the potential to harm indigenous fish populations can accompany ornamental finfish imported into South Korea.

Cervical cancer is the second most common cancer among women worldwide. However, chemotherapies for this cancer often cause many side effects and chemoresistance. Citrus unshiu Markovich peel (CECU) has been used as a traditional medicine for the treatment of various diseases in East Asia. Recently, the anticancer activities and mechanisms of action of CECU extract have been reported in a number of different cancer cell types, but no study has evaluated the therapeutic effect of this natural product on cervical cancer cells. In the current study, the anticancer activity and the underlying molecular mechanism of the chloroform extract of CECU was investigated on HeLa human cervical cancer cells. The results showed that CECU effectively inhibited the proliferation and migration of HeLa cells. Treatment of cells with CECU led to cell cycle arrest at the G2/M phase and activation of extrinsic and intrinsic apoptotic pathways. Furthermore, the proliferation inhibitory effect of CECU was due to the inactivation of AKT and ERK signaling, upregulation of p53 and p21, and downregulation of cyclin B1 and cyclin D1, but not reactive oxygen species (ROS) generation. Furthermore, CECU inhibited the stem‑like features of HeLa cells by downregulating key cancer stemness biomarkers. Therefore, CECU may be an effective complementary and alternative medicine for the prevention and treatment of cervical cancer.

Cervical cancer is the second most common cancer among women worldwide. However, chemotherapies for this cancer often cause many side effects and chemoresistance. Citrus unshiu Markovich peel (CECU) has been used as a traditional medicine for the treatment of various diseases in East Asia. Recently, the anticancer activities and mechanisms of action of CECU extract have been reported in a number of different cancer cell types, but no study has evaluated the therapeutic effect of this natural product on cervical cancer cells. In the current study, the anticancer activity and the underlying molecular mechanism of the chloroform extract of CECU was investigated on HeLa human cervical cancer cells. The results showed that CECU effectively inhibited the proliferation and migration of HeLa cells. Treatment of cells with CECU led to cell cycle arrest at the G2/M phase and activation of extrinsic and intrinsic apoptotic pathways. Furthermore, the proliferation inhibitory effect of CECU was due to the inactivation of AKT and ERK signaling, upregulation of p53 and p21, and downregulation of cyclin B1 and cyclin D1, but not reactive oxygen species (ROS) generation. Furthermore, CECU inhibited the stem-like features of HeLa cells by downregulating key cancer stemness biomarkers. Therefore, CECU may be an effective complementary and alternative medicine for the prevention and treatment of cervical cancer.

The annual rate of ornamental aquatic crustacean importation is increasing globally. The continuous high demand for live ornamental aquatic crustaceans in many countries has also increased the frequency of imported exotic pathogens, which has caused many problems. In the present study, we examined the monthly distribution of viral and bacterial pathogen genes in ornamental aquatic crustaceans imported into South Korea during a 6-month period from February to July 2016. Among the bacterial pathogens identified, Vibrio alginolyticus was detected in cleaner shrimp in June, and Photobacterium damselae was detected in cleaner shrimp in July. Vibrio penaeicida was detected in peppermint shrimp, banded coral shrimp, hinge-beak shrimp, and orange swamp crayfish between February and June. Among the viral pathogens identified, spawner mortality virus was detected in cherry shrimp in May. Finally, hepatopancreatic parvovirus was detected in both cherry shrimp and orange swamp crayfish between February and March. Notably, the genes of two viral pathogens were also detected in cherry shrimp. Although the viruses were not isolated, they are very likely to have been introduced into South Korea and have an influence on Korean aquaculture. In South Korea, HPV is already prevalent and has been reported several times in aquaculture and in the wild; however, SMV infection has not been reported to date. These results demonstrate that many bacterial and viral pathogens are regularly introduced into South Korea via the ornamental aquatic-crustacean trade.

Olive flounder (Paralichthys olivaceus) were fed diets containing 0.01, 0.05, or 0.1% β-1,3-glucan for 30 days, and the effects on general growth, innate immune functions, and mortality caused by pathogen infections were examined. Flounder were found to grow better when fed 0.05 and 0.1% β-1,3-glucan-supplemented diets. The examined innate immunity parameters (lysozyme and complement activities of serum; respiratory burst and phagocytic activities of leucocytes) were all elevated by β-1,3-glucan feeding, although their responsiveness to β-1,3-glucan was not the same. The fish were challenged with three representative pathogens: Vibrio harveyi as a bacterial pathogen, hemorrhagic septicemia virus, and the scuticociliate pathogen Miamiensis avidus. V. harveyi infection-related mortality was significantly reduced by all three β-1,3-glucan concentrations, while mortality due to hemorrhagic septicemia virus infection was significantly reduced only by 0.1% β-1,3-glucan. Moreover, β-1,3-glucan had no effect on M. avidus infection-induced mortality. These results indicate that innate immune functions activated by β-1,3-glucan played a role in reducing bacterial and viral infection-related mortality in olive flounder. In contrast, the same level of immunity was not sufficient to protect against scuticociliatosis-related death. This pronounced activity against bacterial infection indicates that factors other than innate immunity are responsible for resolving bacterial pathogens.

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.

In this study, we determined the effects of clove oil concentration and water temperature on the anesthesia and recovery times, hematological factors, and physiological responses in the olive flounder (Paralichthys olivaceus). To determine these times at three temperatures (17, 21, and 25 °C), fish (mean length [± standard deviation]: 30.0 ± 1.0 cm, mean weight [± standard deviation]: 245.0 ± 27.1 g) were exposed to clove oil at concentrations of 40, 80, 120, 160, 200, 240, and 280 mg L−1. At 0.5, 1, 3, 6, and 24 h, blood samples were collected from 10 fish in the 160 mg L−1 clove oil-treated group (AG) and the non-anesthetic group (NAG) maintained at 21 °C. Subsequently, the following parameters were analyzed: hematocrit and hemoglobin levels; red blood cell count; osmolality; cortisol, glucose, lactic acid, Na+, K+, and Cl- concentrations; and aspartate aminotransferase and alanine aminotransferase activities. Clove oil concentration and temperature exhibited interdependent effects on the anesthesia and recovery times. As the water temperature increased, the anesthesia and recovery times decreased, and as the clove oil concentration increased, the anesthesia time decreased and recovery time was prolonged. AG showed significantly higher plasma cortisol and glucose concentrations than those of NAG at 0.5 and 1 h after anesthesia, but significantly lower concentrations than those of NAG at 24 h after anesthesia. In addition, the lactic acid concentration in AG was lower than that in NAG at 0.5, 1, and 3 h. Intergroup differences for all other blood factors and osmolality were not significant before or after anesthesia. Based on stage III behavior, the anesthesia and recovery times should be less than 180 and 300 s, respectively, to prevent hypoxia in fish under anesthesia. Therefore, the stable anesthesia concentrations for olive flounder (245.0 ± 27.1 g) were 120–160 mg L−1 at 21 °C and 80–240 mg L−1 at 25 °C. Moreover, as 18–21 °C was determined to be the optimal water temperature for olive flounder rearing (˜245.0 g), we suggest that as an optimal anesthetic, clove oil can be optimally used at 160 mg L−1 concentration at 21 °C and that it is a reasonable alternative to other anesthetics. Keywords: Optimal concentration, Recovery time, Hypoxia, Hematological factor

The aim of this study was to assess the treatment potential of quinacrine and oxyclozanide against Microcotyle sebastis (Monogenea: Polyopisthocotylea) infection in cultured black rockfish Sebastes schlegeli. The oral administration of quinacrine led to a reduction in the mean abundance of M. sebastis infection in all quinacrine-treated groups, and the groups of fish administered quinacrine at 50, 100, and 200 mg kg(-1) for 3 consecutive days showed a parasite mean abundance that was 50 to 30% lower compared to that of the control group, suggesting that quinacrine has a therapeutic potential against M. sebastis. Although oxyclozanide showed a very high in vitro killing activity, in oral administration experiments, only the groups of fish administered 200 mg kg(-1) showed less than 50% mean abundance of M. sebastis compared to the control groups, suggesting that the absorption efficiency of orally administered oxyclozanide might be low in black rockfish and/or that M. sebastis might be less sensitive to orally ingested oxyclozanide. As praziquantel has been the sole therapeutic against M. sebastis infection in Korea for a long time, a broadening of available control measures is advisable in order to reduce the possible emergence of praziquantel-resistant M. sebastis. In our study, although quinacrine and oxyclozanide showed a therapeutic potential against M. sebastis, the treatment efficacy was not high enough to replace praziquantel. Thus, after investigations on the pathological effects and pharmacodynamics, use of quinacrine or oxyclozanide in combination with praziquantel may be considered as a way to prevent praziquantel resistance in M. sebastis.

Vibriosis, caused by Vibrio anguillarum , is a common bacterial disease in cultured fish. Treatment of vibriosis can lead to the development of antibiotic resistance or tolerance. Therefore, alternative methods for combatting vibriosis are needed. In this study, we investigated the effects of a subunit vaccine (recombinant flagellin-A [rFlaA]) and adjuvant (CpG-ODN 1668 [CpG]) candidate against V. anguillarum in tilapia ( Oreochromis niloticus ). In an in vivo experiment, fish in the rFlaA + CpG and rFlaA-only groups were first treated with CpG or PBS, followed by vaccination with rFlaA the next day. In the control group, both vaccine and adjuvant were replaced by PBS. The highest survival rate (60%) was observed in fish treated with rFlaA + CpG, compared with 0% in PBS-treated fish or 35% in fish immunized with rFlaA-only. Moreover, sera purified from tilapia treated with rFlaA + CpG exhibited significantly stronger agglutination activity (approximately 3-fold higher) than sera obtained from control fish, whereas sera from fish treated with rFlaA-only showed values 2.5-fold higher than those in control fish. The bacterial survival rates were also lower in sera from tilapia treated with rFlaA + CpG (41%) than in sera from fish treated with rFlaA alone (61%) or control fish (100%). Thus, fish treated with both rFlaA and CpG exhibited the highest immune activation in terms of agglutination and bactericidal activity. In summary, FlaA could be a promising subunit vaccine, and CpG-ODN 1668 could be applied as an adjuvant for immunization against V. anguillarum .

The olive flounder (Paralichthys olivaceus) is an economically important marine fish species in several East Asian countries. Previously, some cell lines have been derived from the olive flounder, which have shown sensitivity to some viral infections that occur in the fish. In this study, we generated a new immortal cell line, named Korea fin Tissue from Sea flounder (KTS), from the fin tissues of olive flounder and successfully subcultured the cells approximately 250 times over 4 years. KTS cells displayed stable growth at temperatures ranging from 25 °C to 31 °C, with optimum growth at 27 °C. Thus, this cell line has a relatively higher and wider range of growth temperatures compared with other reported cell lines. Chromosome analysis showed that the cell line contains 46 chromosomes, which is fewer than those in other cell lines derived from olive flounder. Furthermore, we evaluated the susceptibility of the cell line to infectious viruses such as viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), hirame rhabdovirus (HRRV), and marine birnavirus (MABV). KTS cells showed 100% cytopathic effect (CPE) at 2 weeks. Notably, KTS cells stimulated with CpG-ODN1668 or poly (I:C) showed higher expression of immune genes in the cytoplasm that were recognized by Toll-like receptor 3 (TLR3) or TLR9. These results suggested that the KTS cell line is promising for immune gene expression analysis and other genetic studies. In conclusion, we successfully generated an immortal cell line from the fin tissue of olive flounder. KTS cells have a higher growth temperature range than other reported cell lines, which could prove useful for future cytopathological, genetic, and infectious viral mechanistic studies in this species.

Ethnopharmacology relevance Sparassis crispa, also known as cauliflower mushroom, has been used historically in traditional Asian medicine. It possesses various biological activities, such as immunopotentiation, anti-diabetes, anti-cancer, and anti-inflammatory effects. Recently, we isolated the non-aqueous fraction from methanol extract of S. crispa (SCF4) by using water-organic solvent mixtures and high-performance liquid chromatography (HPLC). In the present study, we identified the anti-inflammatory activity and action mechanism of SCF4 in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophage cells. Materials and methods The chloroform layer isolated from S. crispa methanol extract was separated into seven fractions using preparative HPLC. The fractions were then applied to NO assay to identify the fraction with the best anti-inflammatory activity. The inflammation inhibitory effect and underlying mechanism of SCF4 in LPS-stimulated RAW264.7 cells were assessed using WST-1 assay, enzyme-linked immunosorbent assay (ELISA), ROS assay, and Western blot analysis. Results SCF4 significantly suppressed LPS-induced production of pro-inflammatory mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), and pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)− 6, and IL-1β, without cytotoxicity. In addition, SCF4 downregulated not only the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), but also the activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) stimulated by LPS. SCF4 also blocked the nuclear translocation of NF-κB via reduction of inhibitor of κB alpha (IκBα) degradation. Furthermore, SCF4 inhibited the phosphorylation of transforming growth factor beta-activated kinase 1 (TAK1), an important upstream factor of NF-κB and MAPK signaling mediated through toll-like receptor (TLR). Conclusions These findings demonstrate for the first time the correlation between the anti-inflammatory activity of SCF4 and TLR-mediated NF-κB and MAPK signaling pathways in LPS-stimulated RAW264.7 macrophage cells, suggesting that the non-aqueous extract of S. crispa could be applied as a promising natural product for the prevention and treatment of inflammatory diseases.

Synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) are potent immunostimulators in vertebrates. Based on the backbone structure and the oligonucleotides sequences, CpG-ODNs can be classified into three different classes (A, B, and C) that induce class-specific immune responses. Plasmids with stimulatory CpG motifs in their backbone also show immunostimulatory effects, and a plasmid-based delivery of CpG motifs can be an alternative approach to expensive CpG-ODNs. In this study, we constructed a plasmid (pL-CpGmix) that harbors multiple copies of CpG-ODN 2216 (class A), 1668 (class B), and 2395 (class C) motifs, and evaluated the potential of the constructed plasmid to protect of olive flounder (Paralichthys olivaceus) from viral hemorrhagic septicemia virus (VHSV) and Miamiensis avidus infections. Fish were administered pL-CpGmix, pL-GFP that harbored a green fluorescent protein (GFP) gene, empty vector, or phosphate buffered saline (PBS) alone. On 3 days post-administration, fish were challenged with VHSV or M. avidus. Fish administered pL-CpGmix showed the lowest cumulative mortality (60%) against a VHSV challenge, which was significant compared to that of the control groups (empty vector and PBS). In the M. avidus challenge experiment, fish in the group administered pCpG-mix showed the highest survival rate and serum scuticocidal activity, and those were statistically significant compared to those of control groups. Although fish administered pL-GFP showed lower survival rates than fish administered pL-CpGmix, the GFP-containing vector induced higher survival rates against M. avidus and VHSV infections and higher serum scuticocidal activity compared to the control groups, suggesting that GFP sequence might have an immunostimulating property. The present results showed that the plasmid harboring different classes of CpG motifs enhanced resistance of olive flounder against both M. avidus and VHSV infections.

The phosphoprotein (P) of viral hemorrhagic septicemia virus (VHSV) plays an essential role in viral replication by interconnecting the L protein and the N protein-RNA complex. In this study, to investigate the role of the N-terminal region of the P protein in viral replication, we mutated the first or the first and second or the first, second, and third ATG codon into TGA stop codons. The respective mutants were named P1, P2, and P3. Recombinant VHSVs containing each mutated P gene (rVHSV-P1, -P2, and -P3) were successfully generated by supplying the intact P protein in trans. The rVHSV-P2 and -P3 were not generated from cells expressing truncated P proteins (P1, P2 or P3 protein), but the rVHSV-P1 produced infectious viruses, even in cells without any P-protein-expressing plasmids. Nucleotide sequence analysis of the P gene of rVHSV-P1 showed that a mutation had occurred that resulted in the fourth amino acid (isoleucine, ATT) being changed to methionine (ATG) without a frameshift (P0.5), suggesting that strong selection pressure might facilitate mutations that are advantageous or essential for virus replication. Infectious rVHSV-P2 and -P3 were produced in cells expressing the P0.5 protein, suggesting that the first three amino acids of the P protein of VHSV are dispensable for viral replication. Furthermore, although the P1 protein was shorter than the P0.5 protein by only two amino acid residues, no viruses were produced when the P1 protein was supplied indicating that the fourth and the fifth amino acid residues are indispensable for normal P protein functions involved in viral replication.

The aim of this study was to assess the toxic effects of zinc on the hematological parameters and antioxidant responses in the olive flounder, Paralichthys olivaceus. Juvenile olive flounders (mean length 13.4 ± 1.3 cm, and mean weight 24.3 ± 3.5 g) were exposed to waterborne zinc (Zn) at different concentrations (0, 1, 2, 4, and 8 mg Zn/L) for two weeks. The hematological parameters, hemoglobin level and hematocrit, were substantially decreased. In plasma components, various factors [the inorganic components calcium and magnesium; the organic components glucose and cholesterol; and the enzymatic components glutamic oxalate transaminase (GOT), glutamic pyruvate transaminase (GPT), and alkaline phosphatase (ALP)] were altered by exposure to waterborne zinc, whereas there was no change in the total protein. The responses of the antioxidant superoxide dismutase (SOD) and glutathione S-transferase (GST) were significantly increased, whereas glutathione (GSH) was significantly decreased. The results of this study demonstrate that exposure to waterborne zinc is toxic to P. olivaceus, and induces alterations in the hematological parameters and antioxidant responses. Keywords: Zinc toxicity, Hematological parameters, Plasma components, Oxidative stress, Paralichthys olivaceus

Immunotherapy along with chemotherapy can be a way to enhance therapeutic efficacy and to reduce amount of chemotherapeutics. So, in the present study, we adopted an immunochemotherapy (CpG-ODN 1668 plus doxycycline) to reduce doxycycline dose to be used while at the same time to get the same treatment efficacy against scuticociliatosis in olive flounder ( Paralichthys olivaceus ). The survival rates of fish administered with doxycycline 100 mg/kg plus 10 μg of CpG-ODN 1668 were higher than those of fish administered with doxycycline alone at a dose of 200 mg/kg of fish weight, suggesting that coadministration of CpG-ODN 1668 can reduce doxycycline to half the dose. Furthermore, reduction of CpG-ODN 1668 to 5 μg/fish plus doxycycline 100 mg/kg led to high survival rates that were comparable to doxycycline 200 mg/kg, suggesting that the used amount of both immunotherapeutics and chemotherapeutics can be reduced through coadministration. There have been no reports on the treatment of scuticociliatosis by combining chemotherapy and immunotherapy. In the present study, we demonstrated that a combination of chemotherapy (doxycycline) and immunotherapy (CpG-ODN 1668) was effective against scuticociliatosis in olive flounder fingerlings.

To know the effect of viral hemorrhagic septicemia virus (VHSV) infection on the cellular microRNA expression profile in olive flounder (Paralichthys olivaceus), fish were infected with VHSV, and cellular microRNAs expression was analyzed at 0 (control), 6, 12, 24, 48 and 72 h post-infection (h.p.i.) by the high-throughput sequencing. A total of 372 mature miRNAs were identified, and, among them, 63 miRNAs were differentially expressed during VHSV infection. The differentially expressed microRNAs number was greatly increased from 24 h.p.i. compared to the number at 6 and 12 h.p.i., suggesting that the alteration of microRNAs expression by VHSV infection may be related to the progression of VHSV disease. The target prediction analysis, the GO enrichment analysis, and the KEGG pathway analysis of the predicted target genes showed that various biological pathways could be affected by VHSV infection through the down-regulation or up-regulation of host miRNAs. The present results provide a basic information on the microRNAs related to VHSV infection in olive flounder. Considering broad effects of microRNAs on various biological pathways, data in this study can be used to interpret the mechanism of VHSV pathogenesis, which, vice versa, can be used to develop control measures against VHSV.