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Shrimps infected with WSSV(White Spot Syndrome Virus) generally exhibit white spots in their inner space of carapaces as an acute clinical sign. In an effort to identify the correlation between this acute clinical sign and the condition, the index factors (RNA/DNA concentration and ratio, trypsin activity) were analyzed. A total 580 farmed Fenneropenaeus chinensis and 130 Lithopenaeus vannamei were collected from western and southern fifteen outdoor ponds in Korea. The status of the white spot pathology was divided into four stages (stage 0, stage I, stage II, and stage III), in accordance with the clinical signs as to the size and area of white spots. A significant decrease in RNA concentration and RNA/DNA ratio for multi-infected fleshy prawn (WSSV and vibrio sp.) occurred during the stage III (the whole carapace is covered with a white spot). In particular, RNA/DNA ratio was significantly lower as 1.47±0.04 than other groups. A similar trend was also found in the single infection (WSSV), but the decrease was less than the multi-infection. In the species comparison, both species were vulnerable to the multi-infection, but L. vannamei was more sensitive than F. chinensis (ANOVA, p

Glutathione reductase (GSR) is an enzyme that catalyzes the biochemical conversion of oxidized glutathione (GSSG) into the reduced form (GSH). Since the ratio between the two forms of glutathione (GSH/GSSG) is important for the optimal function of GSH to act as an antioxidant against H2O2, the contribution of GSR as an enzymatic regulatory agent to maintain the proper ratio is essential. Abalones are marine mollusks that frequently encounter environmental factors that can trigger the overproduction of reactive oxygen species (ROS) such as H2O2. Therefore, we conducted the current study to reveal the molecular and functional properties of a GSR homolog in the disk abalone, Haliotis discus discus. The identified cDNA sequence (2325 bp) has a 1356 bp long open reading frame (ORF), coding for a 909 bp long amino acid sequence, which harbors a pyridine nucleotide-disulfide oxidoreductase domain (171–246 aa), a pyridine nucleotide-disulfide oxidoreductase dimerization domain, and a NAD(P)(+)-binding Rossmann fold superfamily signature domain. Four functional residues: the FAD binding site, glutathione binding site, NADPH binding motif, and assembly domain were identified to be conserved among the other species. The recombinant abalone GSR (rAbGSR) exhibited detectable activity in a standard glutathione reductase activity assay. The optimum pH and optimal temperature for the reaction were found to be 7.0 and 50 °C, respectively, while the ionic strength of the medium had no effect. The enzymatic reaction was vastly inhibited by Cu+2 and Cd+2 ions. A considerable effect of cellular protection was detected with a disk diffusion assay conducted with rAbGSR. Moreover, an MTT assay and flow cytometry confirmed the significance of the protective role of rAbGSR in cell function. Furthermore, AbGSR was found to be ubiquitously distributed in different types of abalone tissues. AbGSR mRNA expression was significantly upregulated in response to three immune challenges: Vibrio parahaemolyticus, Listeria monocytogenes, and lipopolysaccharide (LPS), thus indicating its possible involvement in host defense mechanisms during pathogenic infections. Taken together, the results of the current study suggest that AbGSR plays an important role in antioxidant-mediated host defense mechanisms and also provide insights into the immunological contribution of AbGSR.

The viral hemorrhagic septicemia virus (VHSV) is a viral pathogen to cause severe losses in farming industry of olive flounder. Among six-encoding VHSV proteins, NV protein has been shown to have an impact on virulence. Thirteen VHSV strains are isolated from olive flounders showing VHS symptoms and mortality in Korea, and then molecular variation of nucleotide and amino acid sequences of the NV genes were compared. Although common mutations were detected at four sites in nucleotide sequences of NV gene, there was no difference in the amino acid sequences among 13 VHSV variants. We identified substitution of one base of 34 positions within the NV ORF of 13 VHSV isolates and these point mutations resulted in changes in amino acid sequences at 6 residues of NV (8, 56, 81, 88, 117, and 119). Based on finding of NV-mediated decrease of intracellular ATP level, protein expression of six NV variants in flounder cells decreased more than control NV protein. The strongest decreasing activity on cellular ATP level was shown by No. 56 (serine → leucine) of NV variation. The variation changes the hydropathy index significantly. The results imply that we can use appearance of some amino acid variations as virulence markers in VHSV-derived mortality. Statement of relevance 1. Recently VHSV has infected > 80 kinds of fish species. The number of infection host of VHSV is rapidly increased. Among the VHSV-infected fishes, olive flounder is one of the most important fish of aquaculture in Korea, Japan, and China. In every year, we found the massive VHSV-derived mortality of olive flounder aquaculture in Korea. To overcome this VHSV pathogenesis, we need to identify high-pathogenic VHSV appearance at aquaculture in earlier time. 2. Since VHSV gene is highly mutated in time-dependent manner, the efficient vaccine for VHSV-derived disease prevention is not well achieved. Therefore, the other approaches for prevention of epidemics by VHSV should be required. Our research goal is to develop prevention of epidemics propagation of VHSV infection by high pathogenic VHSV prediction with gene variation analysis. 3. In this study, we identified several gene variations of VHSV NV protein from 13 VHSV specimens of severe VHS-showing olive flounders in Korea. The gene variations are associated with amino acid changes of VHSV NV proteins. The amino acid variants of NV protein show the inhibitory activity of fish cellular energy (ATP) generation. The cellular energy deprivation might give a big impact on fish health and mortality. The DNA mutation on specific amino acid changes is possibly applied to develop the PCR diagnosis system for early detection of high pathogenic VHSV appearance. The early pathogenic gene diagnosis of VHSV gives more efficient prevention of epidemics.

A strain of viral haemorrhagic septicaemia virus (VHSV) was isolated from cultured olive flounder (Paralichthys olivaceus) during epizootics in South Korean. This strain showed high mortality to olive flounder in in vivo challenge experiment. The complete genomic RNA sequences were determined and phylogenetic analysis of the amino acid sequences of glycoprotein revealed that this isolate was grouped into genotype IVa of genus Novirhabdovirus. Expression profile of genes in olive flounder was analyzed at day 1 and day3 after infection with this VHSV isolate by using cDNA microarray containing olive flounder 13K cDNA clones. Microarray analysis revealed 785 up-regulated genes and 641 down-regulated genes by at least two-fold in virus-infected fish compared to healthy control groups. Among 785 up-regulated genes, we identified seven immune response-associated genes, including the interferon (IFN)-induced 56-kDa protein (IFI56), suppressor of cytokine signaling 1 (SOCS1), interleukin 8 (IL-8), cluster of differentiation 83 (CD83), α-globin (HBA), VHSV-induced protein-6 (VHSV6), and cluster of differentiation antigen 9 (CD9). Our results confirm previous reports that even virulent strain of VHSV induces expression of genes involved in protective immunity against VHSV.

Infectious hematopoietic necrosis virus (IHNV; n = 18) was identified in the Korean national surveillance program between February 2013 and April 2015, suggesting that IHNV is a major viral pathogen in cultured salmonids. By phylogeny analysis, we found that the JRt-Nagano and JRt-Shizuoka groups could each be further subdivided into three distinct subtypes. The Korean strains were genetically similar to Japanese isolates, suggesting introduction from Japan. Interestingly, the amino acid sequences of the middle glycoprotein gene show that distinct Korean subtypes have circulated, indicating that the settled IHNVs might be evolved stably in cultured salmonid farm environments.

In this study, disease surveillance was performed to monitor the prevalence of viral haemorrhagic septicaemia virus (VHSV) and red seabream iridovirus (RSIV) in olive flounder, Paralichthys olivaceus in 2015. The fish samples were collected in March (60 farms), May (55 farms) and July (52 farms) from different farms in Jeju. Reverse transcription polymerase chain reaction (RT-PCR) (VHSV) or PCR (RSIV) results showed that VHSV detected in 2 farms, but RSIV has not been detected in any farms. The sequences of the nucleocapsid protein (N) and gly-coprotein (G) gene of the 2 VHSV isolates were successfully sequenced. Phylogenetic analysis was included VHSV isolates reported here together with a representative VHSV isolates available in GenBank. Phylogenetic analysis in-dicated that most of Korea VHSV isolates were closely related to the Japan and China genotype Ⅳa which is clearly distinct from the North American genotype Ⅳb.

Rockfish, Sebastes schlegelii (mean weight 14.53 ± 1.14 cm, and mean weight 38.36 ± 3.45 g) were exposed for 4 weeks (2 weeks and 4 weeks) with the different levels of ammonia in the concentrations of 0, 0.1, 0.5, 1.0mg/L at 19 and 24°C. The ammonia exposure induced significant alterations in antioxidant responses. The activities of SOD, CAT, and GST were considerably increased by the ammonia exposure depending on water temperature, whereas the GSH level was notably decreased after 2 and 4 weeks. In the stress indicators, the cortisol and HSP 70 were significantly elevated by the exposure to ammonia depending on water temperature. In innate immune responses, the phagocytosis and lysozyme activity were notably decreased by ammonia exposure depending on water temperature after 2 and 4 weeks. The results suggest that ammonia exposure depending on water temperature can induce the considerable alterations in antioxidant responses, stress, and immune inhibition.

1990년대부터 각종 해수어의 양식이 활발하게 이루어져 각국 에서 종묘 수입이 성해지자 방역체계가 미비한 상태에서 수입 종묘와 같이 질병도 함께 도입되어 우리나라에 없었던 질병들 이 유행하기 시작하였으며(Chun, 2006), 질병의 발병 양상 또 한, 양식 초창기에는 고수온기에 기생충 및 세균에 의한 단독 감 염이 주를 이루었으나 최근에는 수온과 상관없이 연중 다양한 병원체가 혼합감염의 형태로 질병을 일으키고 있어 수산 생물 의 대량 폐사를 유발시키기도 한다(Kim et al., 2006). 최근 들어 외래 질병의 유입 가능성이 증가하고 양식 어류의 질병 연관성 에 대한 관심이 증가하면서 질병을 전반적으로 모니터링하거나 질병과의 상관관계를 구명하고자 하는 연구가 점차 증가하고 있는 추세이다(Cho et al., 2009; Jung et al., 2012; Song et al., 제주의 양식 넙치(Paralichthys olivaceus)를 대상으로 한 여윔증 모니터링(2010-2013)

reported from Pyropia species, with identifications based on culture and genetic analysis of the nuclear internal tran scribed spacer (ITS) region. Recent fungal DNA barcoding studies have shown the low taxonomic resolution of the ITS region and suggested the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene as an alternative molecular marker to identify Pythium species. In this study, we applied an analysis of both the ITS and cox1 regions to clarify the taxonomic relationships of Korean Pythium species. From the results, the two closely related Pythium species (P. chondricola and P. porphyrae) showed the same ITS sequence, while the cox1 marker successfully discriminated P. chondricola from P. porphyrae. This is the first report of the presence of P. chondricola from the infected blade of Pyropia yezoensis in Asia. This finding of the algal pathogen provides important information for identifying and determining the distribution of Pythium species. Further studies are also needed to confirm whether P. chondricola and P. porphyrae are coexisting as algal pathogens of Pyropia species in Korea.

Monthly variations in gametophyte and tetrasporophyte biomass of Chondrus ocellatus Holmes, a commercial carragenophyte alga, were examined at wave-exposed and sheltered shore stations of Jungdori, Wando, Korea from September, 2013 to August, 2014. The frequency of infection of the fronds with a green filamentous endophyte was investigated and the endophyte was identified using tufA analysis. Biomass of C. ocellatus was significantly greater at the exposed shore (331.84 g wet wt. m-2) than at the sheltered shore (181 g wet wt. m-2); the average biomass was 259 g wet wt. m-2. Gametophyte biomass of C. ocellatus accounted for 64.25% of the total biomass (259 g wet wt. m-2); tetrasporophyte biomass was 93.05 g wet wt. m-2 (35.93%). Biomass was minimal in winter and maximal in summer at both stations and similar patterns were found for gametophyte and tetrasporophyte biomass. Frond lengths and weights of C. ocellatus were slightly greater at the exposed shore than at the sheltered shore. Fronds of C. ocellatus were infected by a green endophytic species, which grew in between the cortical and medullar tissue and was identified as Ulvella ramosa by tufA analysis. We conclude that the optimal harvesting period of the C. ocellatus field population in terms of biomass might be autumn, after the rapid growth period. Additional in-depth research on the endophytes, such as infection mechanism and frequency, should be performed in order to maintain and manage the field populations of C. ocellatus.

In this study, we conducted to the development of a rapid antimicrobial susceptibility test method using WST-1 which is known to water-soluble tetrazolium salt, in order to rapidly response against bacterial diseases in fish. Eight of antibiotics which are permissioned for marine organism from government were used to rapid antimicrobial susceptibility testing using the WST-1. As a result, a similar tendency was verified compare to conventional antibiotic susceptibility test results. Generally, the antibiotic susceptibility test method required about 3 days (72 hours) for determine the effective antibiotics, however, we have confirmed that the our method using WST-1 was required at least 36 hours in this study. Consequentially, our method will contribute to development of rapid antimicrobial susceptibility testing for bacterial diseases in fish.

Streptococcus parauberis is recognized as an alpha-hemolytic gram-positive bacterial pathogen associated with bovine udder mastitis. Recently, S. parauberis was identified as the major pathogen of olive flounder (Paralichthys olivaceus) streptococcosis in Northeast Asia, a disease that causes severe economic losses by triggering massive olive flounder mortality. In this study, the complete genome sequence of S. parauberis KCTC11980 was determined and analyzed. S. parauberis KCTC11980 harbors a single chromosome of 2,179,251 bp containing 2281 predicted coding sequences and featuring an average G+C content of 35.65 %. Dot-plot analysis of the entire genomic sequence and phylogenetic analysis of the 16S rRNA gene revealed a close evolutionary relationship between S. parauberis KCTC11980 and S. uberis. Our findings could serve as a basis for understanding the pathogen-host relationship and developing new prophylactic and therapeutic strategies against fish streptococcosis. This study also enhances our understanding of the physiology and evolution of a key representative of the Streptococcaceae family.

The effect of curcumin pretreatment (15-240 μM) in fathead minnow cells infected with viral hemorrhagic septicemia virus (VHSV) was evaluated. Cell viability, apoptosis and viral copy number were analyzed using Cell Counting Kit-8 assay, Annexin V staining, and reverse transcription-PCR, respectively. Pretreatment with 120 μM curcumin showed an increase in viability (>90% of mock) of VHSV-infected cells and reduction in the copy number (0.2-log reduction in VHSV N gene expression), reactive oxygen species and apoptosis in the cells without cytotoxic effects. To understand the mechanisms underlaying the antiviral effects of curcumin pretreatment, a comparative proteomic analysis was performed in four samples (M, mock; C, curcumin-treated; V, VHSV-infected; and CV, curcumin-treated VHSV-infected) in triplicate. In total, 185 proteins were detected. The analysis showed that three proteins, including heat shock cognate 71 (HSC71), actin, alpha cardiac muscle (ACTC1) and elongation factor 1 (EEF1) were differentially expressed between V and CV samples. Network analysis performed by Ingenuity Pathways Analysis (IPA) showed that HSC71 was the primary protein interacting with fibronectin (FN) 1, actins (ACTB, ACTG, F-actin) and gelsolin (GSN) in both V and CV samples and thus is a strong target candidate for the protection from VHSV infection at the viral entry stage. Our proteomics data suggest that curcumin pretreatment inhibits entry of VHSV in cells by downregulating FN1 or upregulating F-actin. For both proteins, HSC71 acts as a binding protein that modulates their functions. Furthermore, consistent with the effect of a heat shock protein inhibitor (KNK437), curcumin downregulated HSC71 expression with increasing viability of VHSV-infected cells and inhibited VHSV replication, suggesting that the downregulation of HSC71 could be responsible for the antiviral activity of curcumin. In conclusion, this study indicates that the suppression of viral entry by rearrangement of the F-actin/G-actin ratio via downregulating HSC71 is a plausible mechanism by which curcumin pretreatment controls the early stages of VHSV infection.

To investigate the causes of emaciation in cultured olive flounder Paralichthys olivaceus in Korea. We performed histological examinations and polymerase chain reaction (PCR) with a new primer set. In most cases, the most severe emaciation was observed in the abdominal area Using PCR on extracted livers, kidneys, spleens, gills, brains, and intestines, we found that areas around the kidneys and intestines were as almost always positive. In significantly emaciated fish, PCR was positive in all internal organs except the gills. In addition, the homology of 812-bp nucleotide sequences of the 28S rRNA gene was more than 99% in emaciated fish. Partial homology with Myxobolus spp. and Cystodiscus axonis, whose data were obtained from GenBank was 86% and 88%, respectively. Histological examinations detected spores in kidneys and intestines but not in other organs. We also performed cohabitation experiments to determine whether infections could be exchanged among species or only within species. Uninfected olive flounder and red sea bream, Pagrus major, cohabitating with emaciated olive flounder showed 100% and 0% cumulative mortality, respectively. Thus the cause of emaciation in cultured olive flounder of Korea is likely due to a new parasite.

Koi herpesvirus (KHV) disease is a lethal disease in common carp, an important food fish in Asian countries, the seed of which is used in restocking programs for freshwater fishery management. We inspected apparently healthy seed stock of common carp Cyprinus carpio L. and Siberian crucian carp Carassius auratus for the presence of KHV using PCR-based diagnostic tests as a part of a stock enhancement program from 2009 to 2010 in Korea. Consequently, KHV was detected from 24 of 232 inspections with yearly detection percentages of 5.2% in 2009 and 15.5% in 2010 using PCR primer sets for TK or SphI-5 as recommended by the OIE Manual of Diagnostic Tests for Aquatic Animals. Results indicate that the SphI-5 primer set was slightly more sensitive than the TK primer set, as shown by a higher detection rate. To determine the genotype of the KHV strains detected in this study, ORF40-specific PCR amplification was conducted, and the PCR products from 6 samples showed 100% nucleotide sequence identity with a Japanese strain (GenBank accession number AP008984) but not with US (DG657948) and Israeli strains (DG177346). This report conclusively demonstrated the presence of KHV in externally healthy seed of common carp and Siberian crucian carp, indicating a possible risk that subclinically infected seed stock can be released with a potential threat to wild populations.

Since the "Aquatic life disease control act" was established in 2009, we have monitored OIE notifiable and legally designated diseases which are associated with white shrimp Litopenaeus vannamei but are as yet outbreak in South Korea. We had monitored only two viral diseases of YHD and IMN, but further added IHHN, TS and WTD in an attempt to reinforce monitoring as a countermeasure against the increasing possibility of imported diseases led by continuous growth in global fisheries trade. We also increased the number of monitoring areas, and shrimp farms. In 2013, we examined a total of 2,650 white shrimp from 29 hatcheries and farms to check whether they were infected with any of the 5 diseases (YHD, IMN, IHHN, TS, WTD). The result showed that none of the samples contained the viruses. To regulation of the exotic diseases from landing in our country and to strengthen prevention, management and control of the diseases on a national level, we must continue the surveillance monitoring of the diseases.

Aquaculture drugs have used to prevent disease in aquaculture field for many years. In spite of many advantages, overdose and abuse may cause environmental pollutions and antibiotic resistances. Many countries try to protect the environment, biospecies and food safety. Recently, Korea enact laws and ordinances such as the Aquatic Animal Disease Control Act. The purpose of this act to contribute to the stable production and control system for aquatic diseases. The Maximum residue limits (MRLs) of aquaculture drugs have been established by Ministry of Food and Drug Safety (MFDS) and Drug licensing was in National Fisheries Research and Development Institute (NFRDI) by Aquatic Animal Disease Control Act. There is 750 items in aquaculture drugs and these are classified into 4 group, which is approval, necessary for prescription, unregulated and safety drug, and banned drug. MRLs of 30 items in aquaculture drug have been established by Food Sanitation Act. Future research is required to determine the suitable for abundant varied fishes of drugs for side effects and safety.

Two species of mitten crab, Eriocheir sinensis and Eriocheir japonica, are commercially valuable in Korea. Millions of their juveniles have been released in attempts to restore ecological habitat and enhance fishery resources. Due to their habitat's singularity, exact species identification should be conducted before juveniles are released. In this study, we used a highly conserved region of the mitochondrial cytochrome b (cyt b) gene to distinguish E. sinensis from E. japonica. We designed a primer set to amplify part of the mitochondrial cyt b gene of both species and obtained 1024 base pairs (bp) of polymerase chain reaction (PCR) product. These PCR products were digested with the restriction enzyme MboII and the species-specific restriction fragment length polymorphism (RFLP) patterns were analyzed. Also, we designed primers for the rapid identification of cyt b gene (498 bp) to distinguish E. sinensis. PCR-RFLP and PCR using new primers are a fast and inexpensive method to access species identification. This simple and rapid method can provide useful information for E. sinensis and E. japonica species identification.

Viral haemorrhagic septicaemia virus (VHSV), the causative agent of viral haemorrhagic septicaemia (VHS), is an epidemic virus of cultured olive flounder Paralichthys olivaceus in Korea. In the present study, the entire glycoprotein (G) gene including several hypervariable regions from 36 isolates of diverse geographic and host origin and 8 Korean VHSV isolates from cultured olive flounder were analyzed. Phylogenetic analysis indicated that most of Asian VHSV belong to the genotype IVa group, suggesting that they originated from a common ancestral virus. Comparative sequence analysis of the complete G protein from Korean VHSV isolates revealed 3 Korean strain-specific nucleotide residues (nucleotide number of G-region: A755, T834 and T1221). These results suggest that Korean VHSV originated from a common ancestor, but these regional specific nucleotide sequences suggest that genetic differences of VHSV are more related to geographic areas than to host fish species.

with four eye spots(E). C, Egg string divided into many capsules on the inner surface of abalone. D, Egg capsule containing developed larvae(L) and nurse eggs(→). SpeciesBodycolorPigmentationOccipital tentacleNotocaetae on chaetiger5Special notochaetae in posterior Palps chaetigersProstomiumInterchaetigersPygidium

The growth, reproduction, gametophyte(G)/tetrasporophyte(T) ratio, and diseases of Chondrus ocellatus populations were examined at 3 sites (Samcheok, Youngduk, Pohang) of East coast and at 2 sites (Hakampo, Manripo) of West coast in between July and August, 2013. Average plant lengths were 6.10~9.69 cm and it was minimum at Manripo and maximum at Pohang population. In general, average plant length and weight of C. ocellatus were greater on East coast than West coast populations. The proportion of vegetative plant was between 26.7~66.7 %, and G/T ratio of total plants including vegetative plants after testing resorcinol method was 3:2 on the East coast where is gametophyte dominant area. However, G/T ratio was 1:1 at Hakampo and 1:2.3 at Manripo representing tetrasporophyte dominance. In the present study, Korean C. ocellatus have various diseases (white colour and green colour), an endophytic alga(Ulvella sp.), and many epiphytic macroalgae and diatoms. Healthy C. ocellatus plants were about 20~40 % in summer population and most of plants had disease. Chondrus ocellatus had a filamentous green alga, endophytic Ulvella sp. which was not identified. The endophyte is easily observed in C. crispus growing in Europe and Canada and it is recognised as a pathogen destroying population and reducing yield of C. crispus. Thus more interest and research on the endophytic algae and disease of C. ocellatus are required.

The impact of global warming on aquatic systems has been a priority research area in the past decade. However, the possibility that increased temperatures will cause shifts in viral disease outbreaks has not been well addressed. In the present study, with increasing water temperature (WT) in the coastal area of Korea, we estimated the possibility of changes in fish viral diseases. From the present time, WT may rise between 0.62 and $1.7^{\circ}C$ by 2050, and the effect on aquaculture could be more adverse than benefitial. Red seabream iridovirus disease (RSIVD) and viral nervous necrosis (VNN) cause high mortality above 22 and $24^{\circ}C$ , respectively, and outbreaks could commence earlier and persist for prolonged periods. Nevertheless, the period of occurrence of viral hemorrhagic septicemia (VHS), which outbreaks at a lower WT ( $18^{\circ}C$ ), could be shorter than the current infectious period. Thermal stress in fish causes reductions in growth and immunocompetence; thus, increases in summer WT can lead to the development of new viral diseases. WT has a strong influence on fish population dynamics; therefore, entry of new viruses and changes in the prevalence of infection can be expected if carrier fishes are introduced or migrate to Korean waters.

Hatchery-reared seeds provides a key source of animal protein for human consumption and restocking for fishery management. For stock enhancement program, we have inspected the hatchery-reared seeds of 33 species in 2009, 44 species in 2010, 43 species in 2011 and 46 species in 2012 for legally designated diseases. Results showed that abalone was the most abundant in the marine species group and then sea cucumber, olive flounder, rockfish and swimming crab were followed. Crucian carp was the most abundant and then mandarin fish, Korean bullhead, melanian snail and Chinese mitten crab were followed in the freshwater species group. The number of inspection for black sea bream, rock bream, scorpionfish, black scraper, and eel has continuously decreased for four years. The inspection for flathead mullet has been carried out only in 2009. The total number of inspection cases for eight pathogens in this study were 8,476 and disqualification cases were 56 (0.67%) by detection of aquatic animals pathogens such as koi herpesvirus, white spot syndrome virus, red sea bream iridovirus or viral haemorrhagic septicemia virus.

Starry flounder, Platichthys stellatus (body length 4.4 ± 0.51 ㎝) that became sick during an outbreak of disease at mariculture facilities at Ulsan, Korea in August of 2012, were examined to identify the cause of the disease. Diseased fish didn't show a unique sign, but the oxidase-positive and gram negative rod was isolated from moribund fish. The bacterium was revealed as Photobacterium damselae subsp. piscicida by biochemical analysis and sequence analysis of the 16S rRNA and capsular polysaccharide (CPS) genes. The isolates (AD5) was carrying susceptible to ofloxacin and gentamycin and showed high growth value at 18 ℃ and 25℃ compared to four other P. damsela strains.

This study aimed to develop an algorithm to determine the optimal injection position in olive flounder, Paralichthys olivaceus when a vaccine is injected into the fish by using a vision‐based automatic equipment measruing the total length (TL), width and weight of the fish. Over a 5‐month period, 500 olive flounders were examined to analyze the relation of the fish size and to the shape and location of its abdomen, using radiography, a ruler plate and scale. There were significant correlations between the TL and the shape and location of the abdomen. The abdomen was located 0.232TL – 2.7221 mm (R2 = 0.8787) from the end of the mouth of the fish. The height and width of the abdomen in the fish were 0.1292TL + 1.8768 mm (R 2 = 0.7935) and 0.183TL–5.9791 mm (R 2 = 0.8641), respectively. The injection point in the abdominal region avoiding organs was determined by calculating the center of gravity of the abdomen volume. This can be expressed as g (0.2759TL – 2.0965, 0.1295TL – 4.2325) on the basis of TL line coordinates. The injection point by the expressed coordinates had errors for the x and y axes as 12.15 mm and 8.28 mm, respectively. These were enough to use the algorithm to injection for the equipment. This automated method to determine the position of any part in the fish can also be used for other purposes, for example, intramuscular injection or auto ‐tagging of fish.

and fish pathogens. Determination of the anti-microbial or anti-viral efficacy of the disinfectant was based on Animal, Plant and Fisheries Quarantine and Inspection Agency Regulation No. 2011-26, Korea. Anti-bacterial efficacy test by broth dilution method was used to determine the lowest effective dilution of the disinfectant following exposure to test bacteria for 30 min at 4℃. Aqua farmsafe and test bacteria or virus were diluted with distilled water (DW), standard hard water (SW) or organic matter dilution (OM) according to treatment condition. Under the our results, disinfectant efficacy of Aqua farmsafe

In 2009, a systemic megalocytivirus infection associated with high mortality was detected for the first time in cultured starry floun der Platichthys stellatus in Korea. Diseased starry flounder had pale bodies and gill coloring and enlarged spleens. Histopathologi cal examinations revealed basophilic enlarged cells in various organs of diseased starry flounder. Polymerase chain reaction (PCR) was performed on tissue samples using three published primer sets developed for the red sea bream iridovirus. PCR products were detected for all primer sets, except 1-F/1-R, which are registered by the World Organization for Animal Health (OIE). The part of the gene corresponding to the full open reading frame encoding the viral major capsid protein (MCP) was amplified by PCR. PCR products of approximately 1,581 bp were cloned, and the nucleotide sequences were analyzed phylogenetically. The MCP gene of the starry flounder iridovirus, designated SFIV0909, was identical to that of the turbot reddish body iridovirus (AB166788).

Cystatins are a well-characterized group of cysteine protease inhibitors, which play crucial roles in physiology and immunity. In the present study, an invertebrate ortholog of cystatin B was identified in Manila clam (Ruditapes philippinarum) (RpCytB) and characterized at the molecular level, demonstrating its inhibitory activity against the well-known cysteine protease, papain. The complete coding sequence of RpCytB (297 bp in length) encodes a 99 amino acid peptide with a calculated molecular mass of 11 kDa and a theoretical isoelectric point of 5.9. The derived peptide was found to harbor typical features of cystatin proteins, including the ‘Q-X-V-X-G’ motif, which was identified as QLVAG in RpCytB. Phylogenetic analysis of RpCytB revealed close evolutionary relationships with its invertebrate counterparts, especially those from mollusks. Recombinant RpCytB (rRpCytB) was overexpressed as a fusion with maltose binding protein (MBP) in Escherichia coli BL21 (DE3) cells. Purified rRpCytB fusion protein exhibited a detectable inhibitory activity against papain, while the control MBP showed an almost constant negligible activity. While quantitative RT-PCR detected ubiquitous RpCytB expression in all tissues examined, the expressions in hemocytes and gills were relatively higher. Upon in vivo immune challenge with lipopolysaccharide (LPS), the expression of RpCytB in gills and hemocytes was down-regulated. Similar challenges with poly I:C and intact Vibrio tapetis bacteria revealed a complicated transcriptional regulation, wherein mRNA expression levels fluctuated over time of exposure. Moreover, a precise induction of RpCytB expression after bacterial infection was detected in gills by in situ hybridization. Collectively, our findings in this study indicate that RpCytB expression is sensitive to host pathological conditions and may contribute cysteine protease inhibitory activity to modulate the immune response.

Enrofloxacin is one of the normally used flouroquinolones in mammalian and fish but its withdrawal time and studies were remain obscure. The residual contents of enrofloxacin in fish muscle were analyzed by using HPLC-FLD. More than 0.1 mg/kg of ENR was detected in muscle tissues and the residues were found over 1 year after treatment. The concentration of ENR in Paralichthys olivaceus was not affected by water temperature and lasted for an extended amount of time. The spike recoveries of ENR in the muscle tissue ranged from 78% to 85%. From this results, we need the prescription by veterinarian or aquatic organism disease inspector in ENR usage to assure safety of fish. Future research is required to determine the recommendation dose of ENR for side effects and safety.

The complement component 8α and 8β are glycoproteins that mediate formation of the membrane attack complex (MAC) on the surface of target cells. Full-length complement C8α ( Rb-C8α ) and C8β ( Rb-C8β ) sequences were identified from a cDNA library of rock bream ( Oplegnathus fasciatus ), and their genomic sequences were obtained by screening and sequencing of a bacterial artificial chromosome (BAC) genomic DNA library of rock bream. The Rb-C8α gene contains 64 bp of 5′-UTR, open reading frame (ORF) of 1794 bp, which encodes a polypeptide of 598 amino acids, 212 bp of 3′-UTR. The Rb-C8β gene contains 5′-UTR of 27 bp, open reading frame (ORF) of 1761 bp, which encodes a polypeptide of 587 amino acids, 3′-UTR of 164 bp. Rb-C8α consists of 11 exons interrupted by 10 introns and Rb-C8β consists of 12 exons interrupted by 11 introns. Sequence analysis revealed that both Rb-C8α and Rb-C8β contain thrombospondin type-1, a low-density lipoprotein receptor domain class A, membrane attack complex/perforin (MACPF) domain and epidermal growth factor like domain. The promoter regions of both genes contain important putative transcription factor binding sites including those for NF-κB, SP-1, C/EBP, AP-1, and OCT-1. Rb-C8α and Rb-C8β showed the highest amino acid identity of 62% and 83% to rainbow trout C8α and Japanese flounder C8β respectively. Quantitative real-time PCR analysis confirmed that Rb-C8α and Rb-C8β were constitutively expressed in all examined tissues, isolated from healthy rock bream, with highest expression occurring in liver. Pathogen challenge, including Edwardsiella tarda , Streptococcus iniae , and rock bream iridovirus led to up regulation of Rb-C8α and Rb-C8β in liver. Positive regulations upon bacterial and viral challenges, and high degree of evolutionary relationship to respective orthologues, confirmed that Rb-C8α and Rb-C8β important immune genes, likely involved in the complement system lytic pathway of rock bream.

In this study, we describe the identification and characterization of manganese superoxide dismutase, an important antioxidant enzyme acting as the chief reactive oxygen species (ROS) scavenger, from rock bream Oplegnathus fasciatus (Of-mMnSOD) at genomic- and transcriptional-levels as well as the biological activity of recombinant protein. The Of-mMnSOD protein portrayed distinct MnSOD family features including signature motifs, metal association sites and the typical active site topology. It was also predicted to be localized in mitochondrial matrix. The Of-mMnSOD had a quinquepartite genome organization encompassing five exons interrupted by four introns. Comparison of its sequence and gene structure with that of other lineages emphasized its strong conservation among different vertebrates. The Of-mMnSOD was ubiquitously transcribed in different rock bream tissues with higher levels in blood cells and metabolically active tissues. Transcription of Of-mMnSOD was kinetically modulated in response to investigational challenges using mitogens (lipopolysaccharide and poly I:C) and live-pathogens (Edwardsiella tarda and rock bream irido virus) in blood cells and liver tissue. The purified recombinant Of-mMnSOD possessed potential antioxidant capacity and actively survived over a range of pH (7.5-11) and temperature (15-40 °C) conditions. Collectively, findings of this study suggest that Of-mMnSOD combats against oxidative stress and cellular damages induced by mitogen/pathogen-mediated inflammation, by detoxifying harmful ROS (O(2)(●-)) in rock bream.

Vaccination by immersion is suitable for mass vaccination of small size fish. However, no viral vaccine has been developed for immersion applications, because of low efficacy. In this study, we evaluated the efficacy and safety of immersion vaccine against viral hemorrhagic septicemia (VHS) containing Montanide IMS 1312 VG adjuvant in olive flounder (Paralichthys olivaceus). Healthy fish were vaccinated by an immersion method with a heat-inactivated FP-VHS2010-1 strain of VHS virus (VHSV) in combination with Montanide IMS 1312 VG for 5 min at 20 ± 2 °C. The control group was vaccinated with sterile PBS. No toxicity of immersion vaccine with Montanide IMS 1312 VG adjuvant was observed by hematological and histopathological analysis. Immersion vaccine with adjuvant enhanced gene expression of immune-associated genes, i.e., genes encoding interleukin (IL)-1β, IL-6, IL-8, and Toll-like receptor (TLR) 3. Relative percent survival (RPS) of fish was measured on weeks 4 and 8 post vaccination. In fish vaccinated with adjuvant, RPS was significantly higher than that of fish vaccinated without adjuvant. The results of the present study provide evidence that the VHSV immersion vaccine with Montanide IMS 1312 VG induces protective immunity in olive flounder against VHS.

Diagnostic monitoring in fish farms with land-based tanks and netpen cases were conducted in eastern, western, southern and Jeju island of Korea during summer of 2007~2011. In total, 2413-fish samples of 4 marine fish species were tested for the detection of bacteria and parasite. Fish species tested were olive flounder (Paralichthys olivaceus), black rockfish (Sebastes schlegeli), red sea bream (Pagrus major), pacific white shrimp (Litopenaeus vannamei). During the diagnostic monitoring from 2007 to 2011, the infection rates by single infection of bacterial or parasitic pathogens were relatively higher than the mixed infections. The main bacterial pathogens in olive flounder, black rockfish and pacific white shrimp were Vibrio spp. (V. harveyi, V. ichthyoenteri, Vibrio sp.). The main bacterial pathogens in red sea bream were also Vibrio sp. and Photobacterium damselae subsp. damselae. The main parasitic pathogens were both Miamiensis avidus and Trichodina sp. in olive flounder, Microcotyle sebastes in black rockfish, Microcotyle tai in red sea bream and Zoothamnium sp. in pacific white shrimp.

(average 300g) at 20±1.0℃ were studied by oral, intramuscular and dipping administration (routes). The concentrations of AM in the plasma were determined by HPLC-UV detector. The average recoveries of AM in spiked samples between 0.01∼10 ppm were ranging from 84.45% to 91.26% for plasma. The limit of detection for AM was 0.05 ppm by using this method. Plasma concentrations of AM were determined after oral dosage (10, 20 and 40 ㎎/㎏ body weight), intramuscular injection (5, 10 and 20 ㎎/㎏ body weight) and dipping (10, 20 and 40 ppm; 1 h). Samples were taken at 1, 3, 5, 10, 15, 24, 30, 48, 96, 144, 216, 264 and 360 h post-administration. In oral dosage of 10, 20 and 40 ㎎/㎏, it’s peak concentrations were 3.62±0.97, 5.20±0.70 and 11.18±0.87 ㎍/㎖, respectively at 10 h post-administration, but AM was not measurable at 144, 360 and 360 h post-administration, respectively. In intramuscular injection of 5, 10 and 20 ㎎/㎏, it’s peak concentrations were 6.92±1.29 ㎍/㎖, 9.89±2.22 ㎍/㎖ and 19.85±2.97 ㎍/㎖, respectively at 5 h post-administration, but AM was not measurable at 216, 264 and 264 h post-administration, respectively. In dipping of 10, 20 and 40 ppm, it’s peak concentrations were 4.39±1.10, 9.57±1.51 and 11.61±1.92 ㎍/㎖, respectively at 3 h post-administration, but AM was not measurable at 264, 264 and 360 h post-administration, respectively. Therefore, the plasma distribution and elimination levels of AM in olive flounder were dosage-dependant manner in all administration routes.Key words

Light and transmission electron microscopy of Batillus cornutus hemocytes revealed differences that the morphological distinctions between blast-like cell, granulocytes and hyalinocytes. Base on the morphological characteristics of the cells, we identified the eight types of hemocytes and present a categorization of the hyalinocytes into six sub-categories. The hemocytes of B. cornutus were observed basophilic cell under the light microscopy. Blast-like cells had a spherical profile with a central nucleus filling almost the whole cell. Granulocytes were characterized by presenting variable numbers of granules. This cell had spherical shape with diameter 7 μm and smooth endoplasmic reticula, granules, mitochondria, glycogen granules in the cytoplasm. Hyalinocytes were the most abundant cell type. Especially, hyalinocyte VI had iirregular an amoebal shape and observed autophagosome and heterophagosome in the cytoplasm. From these results, it is concluded that there are eight types of cells in the hemolymph of B. cornutus. Further studies are now needed to identify the role of these hemocytes in the enzymological and immunological response.

계열의 oxytetracycline과 doxycycline, 그리고 quinolon 계열의 oxolinic acid와 flumequine은 국내 양식장에서 사용되고 있는 항균제이며 (국립수산과학원, 2011) fluoroquinolones 계열의 ciprofloxacin와 norfloxacin은 2009년에 동물에 투여하는 것이 금지되기 전까지 양식 어류의 세균성 질병을 치료하기 위해 사용되었다 (한국동물용의약품판매협회, 2001). 세균성 질병의 치료는 항균제 내성균의 출현 때문에 어려우며 (Aoki and Kitao, 1981) 1980년대 후반부터 국내에서 어류에서 분리된 항균제 내성균에 관해 보고가 있고 (Lee, 1988) 최근에도 항균제 내성률과 다재내성에 관하여 보고되었다 (김 등, 2010). 세균성 질병의 치료를 위한 항균제의 선정은 항균

A 4-week feeding experiment was conducted with juvenile white shrimp Litopenaeus vannamei to compare the growth of shrimp fed different protein sources: fish meal (FM) and soybean meal (SM). By the end of the experiment, the shrimp grew to g (FM) and g (SM). The food and protein conversion rates were significant (P and , respectively. The proximate compositions of the entire body and edible part of the shrimp were similar between FM and SM, except for crude protein. The total amino acid levels were slightly lower in SM than FM, but the difference was not significant. During the experiment, the total ammonia, nitrate, and nitrite concentrations increased continuously in both groups and were slightly higher in SM than FM. In conclusion, soybean meal as a substitute for fish meal results in satisfactory growth and survival of white shrimp.

Hybrid of red seabream (Pagrus major) and Japanese red seabream (Pagrus major) grew rapidly from to August (WT 19.7℃∼24.0℃) while black seabream (Acanthopagrus major) did from September to October (WT 19.9∼23. 0℃). Survival rate of hybrids (85.5%) was a significantly higher than those of the other fish species (20%). Green liver syndrome were observed in black sea bream and Japanease red seabream but did not in hybrids. Microcotyle tai infected 0∼90% in hybrid, 20∼100% in red seabream, 10% in black sea bream and 20∼100% in Japanese red seabream but no significant difference was found among each fish species.

Two viral hemorrhagic septicemia virus (VHSV) isolates, VHSV-KR-CJA and VHSV- KR-YGH, were isolated from viral hemorrhagic septicemia disease outbreaks in flounder farms in South Korea. The VHSV-KR-CJA isolate was isolated from a flounder farm with high mortality (80%), while the VHSV-KR-YGH isolate was isolated from a flounder farm with low mortality (15%), suggesting that these isolates differ in virulence. The virulence of these isolates was eval- uated in juvenile flounder via intraperitoneal injection. Consistent with their virulence in the field, mortality data revealed that the VHSV-KR-CJA isolate was highly pathogenic (cumulative mortal- ity of 80%), while the VHSV-KR-YGH isolate was less pathogenic in flounder (cumulative mortal- ity of 20%). To characterize the genotypes of these viruses, the full open reading frames (ORFs) encoding nucleoprotein N, phosphoprotein P, matrix protein M, glycoprotein G, nonstructural viral protein NV, and polymerase L of these viruses were sequenced and analyzed. Sequence analysis revealed that both isolates are genetically very similar (identical amino acid sequences for P, M, NV, and L and >99.7 and 99.8% amino acid sequence identity for N and G, respectively). Phylogenetic analysis indicated that both of these viruses belong to the Genotype IVa group, sug- gesting that they originated from a common ancestral virus. The low pathogenicity VHSV strain may potentially evolve to become a more pathogenic strain through only a few nucleotide substi- tutions. Further functional analyses of mutations in VHSV genes are necessary to identify factors that determine VHSV pathogenicity in flounder.

The interferon regulatory factor (IRF) members IRF4 and IRF8 contribute to B-lymphocyte development and can act as regulators of immunoglobulin (Ig) light chain gene transcription. These two IRFs are closely interrelated and are expressed at high levels in the lymphoid and myeloid cells of the immune system. In this study, the complete cDNA and genomic sequences of rock bream IRF4 (RbIRF4) and IRF8 (RbIRF8) were identified by homology screening of a multi-tissue normalized cDNA library and a BAC library, respectively, which had been established using Roche 454 GS-FLX™ technology. The full-length RbIRF4 cDNA is composed of 3442 bp and encodes a polypeptide of 462 amino acids; the genomic DNA is 9262 bp in length, consisting of eight exons and seven introns. The full-length RbIRF8 cDNA is composed of 2186 bp and encodes a 422 amino acid polypeptide; the genomic DNA is 4120 bp in length, consisting of nine exons and eight introns. The deduced amino acid sequences of RbIRF4 and RbIRF8 include a conserved DNA-binding domain (DBD) encompassing a tryptophan pentad-repeat and an IRF-association domain (IAD). Several putative transcription factor binding sites were also identified in 5′ flanking region of both RbIRF4 and RbIRF8, and include those of immune-related factors. Quantitative real time PCR analysis of healthy rock bream detected the highest expression levels of RbIRF4 and RbIRF8 in lymphomyeloid-rich tissues. In addition, viral (rock bream iridovirus) and bacterial (Edwardsiella tarda and Streptococcus iniae) infection stimulated RbIRF4 and RbIRF8 expressions in head kidney and spleen. These results suggest not only that RbIRF4 and RbIRF8 may have a protective function against virus and bacteria pathogen invasion in rock bream, but also that IRFs may be immunomodulatory factors of teleost fish.

The eggs of the Pacific oyster, Crassostraea gigas, become infertile when infected by the parasite Marteilioides chungmuensis. Histologically, M. chungmuensis infects the oyster oocyte cytoplasm, and the ovaries take on a "lumpy" appearance once infected, which lowers commercial value of the oyster. This has a negative economic impact on oyster farms in South Korea and Japan. In this study, we compared traditional diagnostic methods (histology) with two molecular-based methods (polymerase chain reaction [PCR] amplification and in situ hybridization [ISH]) to identify M. chungmuensis-infected oysters. The efficacy of PCR and ISH to identify M. chungmuensis-infected oysters was compared to that of routine histology in 100 oysters. Thirty infections were identified using PCR and 16 using histology, whereas 31 infections were identified using ISH. The ISH and PCR assays were more sensitive compared to using histology with standard epidemiological methods. We strongly recommend that early parasitic invasion should be monitored with PCR/ISH methodologies as a basis for developing effective diagnostic techniques to identify M. chungmuensis-infected oysters.