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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

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.

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.

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.

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.

Scuticociliatosis caused by Miamiensis avidus is one of the major diseases in cultured olive flounder ( Paralichthys olivaceus ) in Korea. However, until recently, no effective chemotherapy or vaccine has been developed. Oligodeoxynucleotides containing unmethylated deoxycytidyl-deoxyguanosine motifs (CpG-ODNs) have been shown to be effective in enhancing innate immune responses and preventing infectious diseases in fish. However, it has not been experimented whether CpG-ODNs can be used as a therapeutic tool against infectious diseases in fish. In the present study, we evaluated the therapeutic potential of CpG-ODN 1668 against scuticociliatosis in olive flounder for the first time. The group of fish administered CpG-ODN 1668 at 3 days after the infection showed clearly higher survival rates than the groups of fish administered GpC-ODN 1720 or PBS, indicating a high therapeutic potential of CpG-ODN 1668 against scuticociliatosis. However, the treatment efficacy of CpG-ODN 1668 was diminished when administered at 6 days post-infection, and no treatment effect was found when administered at 9 days after the infection. These results suggest that early diagnosis through continuous monitoring and early treatment with CpG-ODN 1668 are essential for increase of treatment efficacy. In conclusion, the present study demonstrated for the first time the possible use of CpG-ODN 1668 in treatment of scuticociliatosis in olive flounder.

Protection of olive flounder ( Paralichthys olivaceus ) from virulent Edwardsiella tarda infection by oral immunization with two auxotrophic genes – alanine racemase gene ( alr ) and aspartate semialdehyde dehydrogenase ( asd ) gene – knock-out E. tarda mutant ( ΔalrΔasd E. tarda ) was previously demonstrated. In the present study, we investigated the effects of cimetidine, a histamine 2 (H 2 ) receptor antagonist, on the arrival of the live auxotrophic E. tarda mutant to the intestine of olive flounder and on the protective efficacy of the live ΔalrΔasd E. tarda oral vaccine. In the experiment to determine optimal administration time of cimetidine, fish administered cimetidine at 3 h before the administration of the E. tarda mutant showed the highest bacterial colony number from the intestine. In the experiment to determine optimal dose of cimetidine, the groups of fish administered 100 mg or 200 mg of cimetidine per kg of fish showed significantly higher CFU numbers in the intestine compared to the group intubated with cimetidine 10 mg or 50 mg per kg of fish. However, as there was no significant difference between cimetidine 100 mg and 200 mg groups, a dose of 100 mg per kg of fish was chosen as the optimal dose of cimetidine. In the oral vaccine efficacy experiment, the group of fish administered the live auxotrophic E. tarda mutant in combination with the above optimal regime of cimetidine showed the highest serum agglutination activity and survival rates, suggesting that cimetidine can be used to enhance the protective efficacy of the oral live bacterial vaccines in fish. To our knowledge, this is the first study that demonstrated cimetidine-mediated enhancement of protective efficacy in oral vaccine of fish.

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.

Scuticociliatosis is a leading cause of mass mortality in cultured olive flounder (Paralichthys olivaceus) in Korea. However, still no effective treatment measures have been developed. In the present study, we selected two drugs, doxycycline and albendazole, that were demonstrated to have high in vitro scuticocidal activity and possess ability to cross blood–brain barrier (BBB), and evaluated their treatment efficacy against scuticociliatosis caused by Miamiensis avidus in olive flounder. In both oral and bath treatment experiments, groups of fish fed a diet containing doxycycline or bathed with freshwater containing doxycycline showed clearly higher survival rates than control groups. Although the groups of fish fed a diet containing albendazole or bathed with freshwater containing albendazole showed higher survival rates than the control group, the rates were greatly lower than those of the groups treated with doxycycline. Poor intestinal absorption, low solubility in water, and low lipophilicity of albendazole were considered as the possible cause of the low efficacy. In conclusion, the present results suggest that doxycycline can be used to treat systemic scuticociliatosis in olive flounder through oral administration and bath.

The objective of this study was to investigate the effects of different classes of CpG oligodeoxynucleotides (CpG-ODNs) on resistance of olive flounder ( Paralichthys olivaceus ) against viral hemorrhagic septicemia virus (VHSV) and Miamiensis avidus infections. Fish administered CpG-ODN 2216 (A-class ODN) showed the highest resistance against VHSV infection and the strongest expression of Mx and ISG15 genes. Fish administered CpG-ODN 2395 (C-class ODN) showed intermediate resistance against VHSV between ODN 1668 and ODN 2216. Whereas, olive flounder intraperitoneally (i.p.) injected with CpG-ODN 1668 (B-class ODN) showed no enhanced resistance against VHSV challenge and no significant increase in expression of Mx and ISG15 genes. In contrast, in the M. avidus challenge experiment, fish injected with CpG-ODN 1668 showed the highest survival rate and the strongest scuticocidal activity of serum. Similar to the results in the VHSV infection experiment, administration of CpG-ODN 2395 elicited secondly higher survival rate against M. avidus challenge. Although fish administered CpG-ODN 2216 eventually showed 100% mortality by M. avidus challenge, the cumulative mortality until 2 weeks post-challenge was similar to the group of fish injected with CpG-ODN 1668. The present results suggest that CpG-ODNs belonging to different classes have different abilities to increase resistance against different pathogens in olive flounder. Therefore, selection of a CpG-ODN appropriate to the characteristics of a certain pathogen is crucial to induce immune responses that are optimal for defense against the pathogen.