Fish larvae are immediately exposed to microbes from hatching to maturation of their lymphoid organs, therefore effective innate mechanisms is very important for survival. However, the knowledge of the development of immune system in fish is limited and in demand now. In vertebrates, recombination-activating gene 1 (RAG-1) and immunoglobulin M (IgM) have been considered as very useful markers of the physiological maturity of the immune system. In this study, the expression of the both genes was assessed throughout the early developmental stages of olive flounder larvae (5-55 dph) and used as markers to follow the development of immune system. RAG-1 and IgM mRNA expression was detectable at 5 dph and remained so until 55 dph. These patterns of expression may suggest that the olive flounder start to develop its function around 5 dph. Tissue distribution was found that both genes mRNAs are only expressed in the immune-related organ such as spleen, kidney and gill. The early detection of IgM mRNA led to the investigation of its presence in oocytes. Both RAG-1 and IgM mRNA transcripts were detected in unfertilized oocytes, suggesting that they are maternally transferred. The biological significance of such a phenomenon remains to be investigated.

In Pleuronectiformes, blind-side malpigmentation (hypermelanosis) is common in cultured flatfishes, and is economically important. To understand the mechanism of blind-side hypermelanosis in flatfishes, we examined when the malpigmentation initially occurred, and studied how the symptoms proceeded during early development of the starry flounder, Platichthys stellatus. To assess quantitative pattern changes of blind-side skin, we observed morphological development of the whole body from 22 (total length [TL] 10.0±0.2 mm and body weight [BW] 8.8±0.57 mg) to 110 days (TL 23.4±0.7 mm, BW 193.6±23.3 mg) after hatching (DAH), and also examined the malpigmented area rate of blind-side skin and the malpigmented fish ratios. The experimental animals were reared in fiberglass-reinforced plastic (FRP) tanks in water at a temperature of 18.9±1.9℃ and salinity of 32.6±0.6 psu and were fed with rotifer and Artemia nauplii from 22 to 48 DAH, and with A. nauplii and commercial feed from 49 to 110 DAH. As results, the first staining patch seen by the naked eye was observed around the area between the anus and pelvic fin or caudal edge of the trunk at 80 DAH (TL 20.6±0.5 mm, BW 112.5±8.8 mg). The pigmented area and the pigmented fish ratios were significantly increased from 80 to 110 DAH. These results indicated that malpigmentation on the blind side of starry flounder was initially observed at about 2 cm in length and 100 mg in weight, and the pigmented domain on the blind-side skin was continually broadened by the differentiation of pigmented cells (melanophores and xanthophores) with growth.

In this study, oocyte steroidogenesis are investigated in relation to oocyte development in the starry flounder, Platichthys stellatus, a marine multiple spawner. Vitellogenic (0.52 and 0.55 mm oocyte diameter) and mature oocytes (0.63, 0.66 and 0.71 mm oocyte diameter) were incubated in vitro in the presence of [3H]17α-hydroxyprogesterone ([3H]17α- OHP) as a precursor. Steroid metabolites were extracted from the incubated media and oocytes, the extracts were separated and identified by thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatographymass spectrometry (GC-MS). The major metabolites produced from [3H]17α-OHP were androgens [androstenedione (A4) and testosterone (T)] and estrogens [17β-estradiol (E2) and estrone (E1)] and progestins [17α,20α-dihydroxy-4-pregnen- 3-one (17α20αP) and 17α,20β-dihydroxy-4-pregnen-3-one (17α20βP)] in vitellogenic and mature oocytes. The results from this study suggest the potential roles of E1 in the oocytes with diameter 0.52-0.71 mm, 17α20αP and 17α20βP at the oocytes of 0.63, 0.66 and 0.71 mm.

TCR subunits are members of membrane-bound receptors which allow the fast and efficient elimination of the specific fish pathogens have regulated function in adaptive immunity. Sequence structure of TCR subunits have been reported for various teleosts, but the information of each TCR subunit functional characterization through expression analysis in fish was unknown. In this study, we examined the gene expression of TCR subunits in the early developmental stages and observed transcript levels in various tissues from healthy adult olive flounder by RT-PCR. The mRNA expression of alpha subunit was already detected in the previous hatching step. But the transcripts of another TCR subunit were not observed during embryo development and increased after hatching and maintained until metamorphosis at the same level. It was found that all TCR subunits mRNAs are commonly expressed in the immune-related organ such as spleen, kidney and gill, also weak expressed in fin and eye. TCR alpha and beta subunit were expressed in brain, whereas gamma and delta were not expressed same tissue. The sequence alignment analysis shows that there are more than 80% sequence homology between TCR subunits. Because it has a high similarity of amino acid sequence to expect similar in function, but expression analysis show that will have may functional diversity due to different time and place of expression.

The innate immune system is the only defense weapon that invertebrates have, and it is the fundamental defense mechanism for fish. The innate immune response is important in newly hatched flounders because it is closely involved in the initial feeding phase, which is why it is essential for survival during the juvenile period. The expression analysis of genes involved in the innate immune response in the olive flounder (Paralichthys olivaceus) in the days after hatching is incomplete. Therefore, we have begun to examine the expression patterns of genes specifically induced during the development of the innate immune system in newly hatched flounders. Microscopic observation showed that pronephron formation corresponded with the expression of perforin-encoding gene. These results suggest that perforin plays a vital role in the innate immunity of the kidney during developmental stages. Perforin expression was strong at the start of the development of the innate immune response, and continued throughout all the development stages. Our findings have important implications with respect to perforin’s biological role and the evolution of the first defense mechanisms in olive flounder. Further studies are required to elucidate the perforin-mediated innate immunity response and to decipher the functional role of perforin in developmental stages.

Fish larvae are immediately exposed to microbes from hatching to maturation of their lymphoid organs, therefore effective innate mechanisms is very important for survival in such an environment. The key component of innate immune system, C3 is central protein of all activation pathways of the complement system, leading to inflammatory reactions, such as opsonisation, chemotaxis, and cell lysis of pathogens. Although, innate mechanisms is essential for survival in the early stage of development, little is known about defence mechanisms. In this study, the alignment analysis showed that amino acid sequence of C3 from olive flounder liver EST homologous to other known C3 sequences with 73-99% identity. Also, we examined the tissue distribution of olive flounder C3 and analyzed expression pattern from the fertilized egg until 28 days post hatching. As a result, olive flounder C3 mRNA was expressed only in the liver and the mRNA level more increased as developmental proceed during the early stage. These results may suggest that olive flounder C3 plays an important function in the early immune response of olive flounder larvae.

To effects of sex maturation in olive flounder by regulating long photoperiod, gonadal development and GTH mRNA expression in the pituitary were investigated. Photoperiod was treated natural photoperiod and long photoperiod (15L:9D) conditions from September 2011 to March 2012. The results showed that natural photoperiodic group showed a higher gonadosomatic index (GSI) than long photoperiodic group during the spawning season (March 2012). The histological analysis of ovarian tissue showed that natural photoperiod group of ovaries contained vitellogenic oocytes, but long photoperiod group of ovaries mainly contained perinucleolus staged oocyte and oil-drop staged oocytes. The FSH mRNA of olive flounder, under natural photoperiod group, showed a significantly higher expression but no significant difference under long photoperiod group. The LHβ mRNA showed a significantly higher expression only under natural photoperiod group. These results may suggest that long photoperiodic information regulates secretion of pituitary FSH and LH and maintain early growing stage of gonadal development in this species.

Olive flounder (Paralichthys olivaceus) is one of the commercial important flatfish species in Korea. The ocular signal transduction pathway is important in newly hatched flounders because it is closely involved in the initial feeding phase thus essential for survival during the juvenile period. However, the study of gene expression during ocular development is incomplete in olive flounder. Therefore we examined the expression analysis of specifically induced genes during the development of the visual system in newly hatched flounders. We searched ocular development-involved gene in the database of expressed sequence tags (ESTs) from olive flounder eye and this gene similar to arrestin with a partial sequence homology. Microscopic observation of retinal formation corresponded with the time of expression of the arrestin gene in the developmental stage. These results suggest that arrestin plays a vital role in the visual signal transduction pathway of the retina during ocular development. The expression of arrestin was strong in the ocular system during the entirety of the development stages. Our findings regarding arrestin have important implications with respect to its biological role and evolution of G-protein coupled receptor (GPCR) signaling in olive flounder. Further studies are required on the GPCR-mediated signaling pathway and to decipher the functional role of arrestin.

Cathepsins are members of the multigene family of lysosomal cysteine proteinases and have regulated function in several life processes. The potential role of cathepsin F cysteine gene was expected as protease in the yolk processing mechanism during early developmental stage, but expression analysis was unknown after fertilization. The alignment analysis showed that amino acid sequence of cathepsin F from olive flounder liver expressed sequence tag (EST) homologous to cathepsin F of other known cathepsin F sequences with 87－98% identity. In this study, we examined the gene expression analysis of cathepsin F in various tissues at variety age flounder. Tissue distribution of the cathepsin F mRNA has been shown to be ubiquitous and constitutive pattern regardless of age in each group, although derived from cDNA library using liver sample. The mRNA level of cathepsin F more increased as developmental proceed during embryogenesis and early developmental stage, especially increased in the blastula, hatching stage and 3 days post hatching (dph). As a result, it may suggest that the proteolysis of yolk proteins (YPs) has been implicated as a mechanism for nutrient supply during early larval stages in olive flounder.

Olive flounder (Paralichthys olivaceus) is a most important aquaculture species in Korea. Like most marine fishes, olive flounders are stomachless at first feeding and aquired gastric function during the metamorphosis, so food was mainly digested by pancreatic enzyme from first feeding to premetamorphosis. However, comprehensive analysis of pancreatic and gastric digestive enzyme of olive flounder at early developmental period is still unclear. In the expression study of pancreatic and gastric digestive enzyme by real-time PCR at early developmental stage, pancreatic enzyme such as chymotrypsinogen 2, preproelastase 2 and 4, pancreatic protein somatomedin-B domain (PPSB) mRNA expression were initiated at first feeding and strongly expressed in the pancreas developmental stage, while gastric digestive enzyme signal was not at all detected during same period. Although, trypsinogens were secreted from pancreas and have similar amino acid sequence, trypsinogen 3 expression induction was detected both pancreas and stomach developmental stage, while trypsinogen 2 expression was significantly increased only post-metamorphosis period. Pepsinogen mRNA expression was only detected at metamorphosis according to stomach differentiation. Lipid digestive enzyme, lipase and intestine fatty acid binding protein 1 (I-FABP 1), were already reached a certain level at beginning of hatching and more increased during early developmental stage and then gradually decreased before metamorphosis. These results suggested that feed ingestion of olive flounder was exclusive charged by pancreatic digestive enyme, lipid digestive enzyme and trypsinogen 3 from first feeding and then fully swiched by gastric digestive enzyme and trypsinogen 2 from metamorphosis period.

This study was conducted to analyze the difference of body types within and between wild and farmed populations of olive flounder Paralichthys olivaceus using measured records of morphological traits. The results showed that surveyed traits and standard deviation were of body weight, of total length, and of body length. Also body height, body shape index and condition factor were , and , respectively. As result of least squares mean and standard error for each trait assumed in this study, those of farmed population showed significantly higher than those of wild population in all traits, exclusively in total length and body length (p<0.01). Particularly, the values of the body height and the body weight of the farmed population were higher than those of the wild population in the same total length. And the phenotypic correlation coefficients of the body weight, the total length, the body length and the body height showed strong positive correlation in all populations. These result suggested that morphological differences exist in farmed and wild flounder. Therefore, introduction of wild flounder is essential for the future production to improve the body type of farmed flounder, and parental fish should be chosen by considering selection of commercially important traits in the production process.

Olive flounder Paralichthys olivaceus is one of the most widely cultured fish species in Korea. Although olive flounder receive attention from aquaculture and fisheries and extensive research has been conducted eye morphological change in metamorphosis, but little information was known to molecular mechanism and gene expression of eye development- related genes during the early part of eye formation period. For the reason of eyesight is the most important sense in flounder larvae to search prey, the screening and identification of expressed genes in the eye will provide useful insight into the molecular regulation mechanism of eye development in olive flounder. Through the search of an olive flounder DNA database of expressed sequence tags (EST), we found a partial sequence that was similar to crystallin beta A1 and gamma S. Microscopic observation of retinal formation correspond with the time of expression of the crystallin beta A1 and gamma S gene in the developmental stage, these result suggesting that beta A1 and gamma S play a vital role in the remodeling of the retina during eye development. The expression of crystallin beta A1 and gamma S were obviously strong in eye at all tested developing stage, it is also hypothesized that crystallin acts as a molecular chaperone to prevent protein aggregation during maturation and aging in the eye.

강도다리, Platichthys stellatus는 가자미목 가자미과 어류로서, 최근 어류양식 산업에서 넙치의 대체어종으로 주목받고 있다. 그러나 양식과정 중 나타나는 이 종의 무안측에서 발현되는 흑화 현상(blind-side hypermelanosis)은 많은 경제적 손실을 초래하고 있다. 이 현상은 자연계에서는 극히 일부분만이 관찰되지만 양식을 할 경우, 초기 발달단계에서 높은 비율로 발현된다. 그러나 아직까지 그 원인이 정확하게 무엇인지 밝혀지지 않고 있다. 단지 자연계와 다른 고밀도의 양식사육 조건이 만들어내는 스트레스가 이 이상 발현의 원인이 가능성에 주목되고 있다. 이에 본 연구에서는 밀도가 강도다리의 blind-side hypermelanosis 발현에 미치는 영향을 파악해 보았다. 실험어는 전장 3.2±3.4 cm, 체중 0.6±0.2 g의 강도다리 치어를 사용하였고, 100 L 유리수조에 수용량 80 L에 100개체와 400개체의 두 실험구를 설정하여 120일간 실험을 실시하였다. 평균 수온 21.9±5.3℃, pH 7.6±0.1, DO 6.1±1.7 mg/L 및 염분도는 28.3±1.3 psu였다. 사료공급 후에는 기록된 사료 공급량을 바탕으로 일간 개체당 사료섭식량(daily food Intake: DFI, mg/fish/day) 및 사료효율(feed efficiency: FE, %)을 구하였다. 실험구별 무안측 착색률을 조사하기 위해 실험 개시 시 평균 착색률(8.84±3.84%)을 제시하고, 실험개시 후 60일, 120일에 실험구별 강도다리 30마리씩 2반복으로 2-phenoxyethanol로 마취시킨 후, 10% 포르말린에 고정시켰다. 이후 무안측면을 디지털 카메라로 촬영한 뒤 컴퓨터 영상분석시스템(QWIN V3 Leica, Germany)을 통해 착색반문의 면적률(=착색반문 표면적/무안측 총면적×100%)을 구하였다. 또한 측정된 개체별 무안측 착색 면적률을 바탕으로 1% 이상 무안측 흑화율을 보인 개체를 정상으로 구분하여, 실험구별 흑화 빈도를 계산하였다. DFI는 저밀도구(100 inds./80 L)가 고밀도구(400 inds./80 L) 보다 높게 나타났고, 반면 FE는 고밀도(189.0±3.6%)가 저밀도(181.2±5.8) 보다 높은 값을 나타내었다. 생존율은 큰 차이를 보이지 않았다. 성장에 있어 전장은 밀도구별 차이를 나타내지 않았지만, 체중에 있어서는 고밀도 보다 저밀도에서 보다 빠른 성장을 보였다. 군집내 흑화개체비는 성장에 따라 두 실험구 모두 유의하게 증가하였지만, 밀도구별 차이는 찾아 볼 수 없었고, 흑화면적률 역시 동일한 경향을 나타내었다. 따라서 blind-side hypermelanosis에 있어 밀도는 주요인이 아니라는 것을 의미하며, 다른 환경인자 또는 유전적 요소가 핵심적인 유발 인자일 가능성이 높음으로 이에 대한 지속적인 연구가 요구된다.

To investigate the involvement of prepro-proopiomelanocortins (POMCs) with hypermelanosi on the blind side of olive flounder, Paralichthys olivaceus, we surveyed tissue sites in which the three POMCs genes are expressed, and compared pituitary-ofPOMCs (ofPOMC-Ⅰ, Ⅱ and Ⅲ) mRNA activities between the normal and the hypermelano flounders. For the studies, we refereed to the information of ofPOMC-Ⅰ and Ⅱ genes in NCBI, but we try to newly discover ofPOMC-Ⅲ sequence. At first, we cloned and characterized cDNA encoding its ofPOMC-Ⅲ from the pituitary of olive flounder The ofPOMC-Ⅲ cDNA consists of 1,258 bp nucleotides and 7 peptides (Signal peptide, N-POMC, α-MSH, CLIP, N-β-LPH, β-MSH and β-END-like peptide) encoded in 231 amino acids. In the test of tissue distribution of ofPOMCs in olive flounder, we found that ofPOMC-Ⅰ is expressed in pitutary only, but ofPOMC-Ⅱ and Ⅲ are expressed in all tissues examined. In the comparison of ofPOMCs mRNA expression between the normal and the hypermelano fish, expression of ofPOMC-Ⅰ, Ⅱ mRNA was significantly higher in the hypermelano group than in normal group, but ofPOMC-Ⅲ was not significantly different between two groups.

Foodfish species에서 어류의 성장과 관련이 있는 체중과 전장은 경제적으로 중요한 형질이다. 어류의 성장에 관여하는 유전자를 확보하기 위하여 동일한 사육환경에서 체중이 2배 이상 차이를 나타낸 강도다리를 선별하여 유전자 발현양상을 조사하였다. 강도다리의 체중을 기준으로 large 그룹과 small 그룹으로 나누었으며, ACP(annealing control primer) system을 이용하여 근육조직에서 발현량 차이를 나타내는 차등발현유전자 5개를 얻었다. 5개의 차등발현유전자 중 1개는 creatin kinase muscle type(CKM1)에 해당하는 유전자 단편이었고, 4개는 unknown gene이었다. CKM1 유전자는 성장이 빠른 강도다리 그룹에서 발현량이 4.15배 많았으며, 전체유전자(genomic structure)는 8개의 exon과 7개의 intron으로 이루어져 있었다. CKM1 유전자는 근육조직을 비롯하여 신장, 아가미, 지느러미와 혈액에서는 발현되었으나, 간 조직에서는 발현되지 않았다.

This expe끼ment was conducted to evaluate the effect of two different organic acid products as antibiotic replacement in olive flounder paralichthys olivaceus . Fish averaging 3.5 ::!:: 0.05 g(mean ::!:: SD) were fed one of the ten semi-purified diets : Diet 1 ; Control , Diet 2 ; add antibiotics - 50mg OTC / kg body weight / day(OTC), Diet 3; Add organic acid bland A(OABA) - 4g/kg diet, Diet 4 ; add organic acid bland B(OABB) - 4g/kg diet for 10 weeks. Total gut microflora counts were significantly higher in the control group compared to the OTC and organic acid groups(P <0.05). Fish fed OABA, 。ABB and OTC had lower gut Vibrio counts compared to the control, but were not significantly different. Results from the challenge study indicate that mortality in the different treatment groups (50%) was significantly lower than those observed for the control group (100%). There were no differences in mortality between the OTC and organic acid groups. Overall findings from this study indicate that the organic acid blends A and B were as effective as oxytetracycline, an antibiotic, in regulating total gut bacterial numbers, Vibrio counts and providing protection against a pathogen such as Edwardsiella tarda.

배합사료를 공급한 양식 넙치와 생사료를 공급한 양식넙치의 핵산 관련 물질 및 유리아미노산 측정을 통한 품질의 차이를 비교하였다. 넙치 등근육의 핵산관련물질은 모든 실험사료구에서 공통적으로 AMP 함량이 가장 높았고 그 다음으로 IMP의 함량이 높았다. 공급한 사료에 따라서 HxR의 성분에는 유의적인 차이(p

본 연구에서는 단백질(51~5%) 및 지질(9~15%) 함량이 다른 배합사료(EP)와 생사료(MP)를 공급하면서 사육한 양식넙치의 육질을 비교해 보고자 하였다. 사육실험은평균체 중 106g의 넙치를 원형수조에 40마리씩 실험구별로 2반복으로 수용하면서, 단백질 및 지질 함량이 서로 다른 실험 배합사료(EP1, EP2, EP3)와 수입산 상품 배합사료(EP4) 및 MP를 16주간 공급하면서 사육하였다. 수분함량은 생사료(MP)를 공급한 넙치의 등근

In the present study, to understand the process of abnormal pigmentation (hypermelanosis) on the blind side of olive flounder P. olivaceus during early development, we investigated an time that staining on the blind side is initially observed with the naked eyes, counted the number of stained spot on the blind, and calculated the ratio of area stained on the blind side, and the ratio of fish pigmented on the blind side with growth. The present experiment was initiated at 24-day after hatching (dah), just after the starting of metamorphosis, in which fish mean size is total length (TL) 12.4±0.2 ㎜ and body weight (BW) 19.9±0.7 ㎎, and was finalized at 94-dah (TL 72.0±0.6 ㎜, BW 3,912.3±119.4 ㎎). The experimental animals were reared in FRP tank at 20±0.5℃ in water temperature and 30.9±0.1 psu. The experimental animals were feed with rotifer, Artemia nauplii and commercial food according growth stage. As results, the pigmented spots are composed with adult-type melanophores. The first spot was found in microscopy level at 38-dah (TL 18.3±0.4 ㎜, BW 57.6±4.1 ㎎), but the first staining spot observed with the naked eye was found 52-dah (TL 37.6±0.5 ㎜, BW 534.2±30.9 ㎎) around the pectoral fin and the caudal peduncle. The pigmented spots became more distinctive and expanded around the edge of rear on the blind side. The number of spots and the ratio of area stained on the blind side sharply increased between 31-dah and 80-dah, and between 66-dah and 94-dah, respectively. The ratio of fishes stained on the blind side increased between 45-dah and 73-dah, and then was stagnated to 94-dah. Therefore, these results concluded that the pigmentation on the blind side of olive flounder is not an temporary and reversible color change but an irreversible abnormal phenomenon that pigment cells is continuously differentiated on the blind side with growth for early development just after metamorphosis in artificial culture conditions.