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To develop a promoter capable of driving transgene expression in non-model fish, we identified and characterized the muscle-specific alpha-actin gene in olive flounder, Paralichthys olivaceus (PoACTC1). The regulatory region of PoACTC1 includes putative regulatory elements such as a TATA box, two MyoD binding sites, three CArG boxes, and a CCAAT box. Microinjection experiments demonstrated that the regulatory region of PoACTC1, covering from -2,126 bp to +751 bp, just prior to the start codon, drove the expression of red fluorescent protein in developing zebrafish embryos and hatching olive flounder. These results suggest that the regulatory region of PoACTC1 may be useful in developing a promoter for biotechnological applications such as transgene expression in olive flounder.

The olive flounder (Paralichthys olivaceus) is one of the most important aquaculture species in Korea. Previous studies focused on the morphological changes and only analysed digestive enzyme expression patterns of certain digestive systems. A comprehensive analysis of the pancreatic and gastric digestive enzymes of the olive flounder in its early developmental stages has not been performed. In this study, we determined the mRNA expression levels of pancreas and stomach digestive enzymes by extracting total RNA from the whole body of olive flounders from hatching to post-metamorphosis and conducting real-time PCR analysis with gene-specific primers for each isoform. The pancreatic digestive enzymes were initially and strongly expressed from the first feeding period to pre-metamorphosis. The expression of trypsinogen 3 was detected from the early stage of development to metamorphosis, whereas trypsinogen 2 was significantly increased only in the post-metamorphosis period. The mRNA expression of pepsinogen was only detected at metamorphosis and corresponded to stomach differentiation. The lipid digestive-related enzymes had already reached a certain level at the beginning of hatching, then increased during the early developmental stage and gradually decreased before metamorphosis. These results suggest that food ingestion is exclusively digested by pancreatic digestive enzymes and trypsinogen 3 during the early developmental stage until pre-metamorphosis, and it then fully switches to digestion by pepsinogen and trypsinogen 2 in mid-metamorphosis. In conclusion, understanding the expression profiles of digestive enzymes in the pancreas and stomach during the early developmental stages is an important consideration when developing formulated larval diets for aquacultured olive flounder.

반수체(haploid)는 세포당 하나의 유전체 또는 염색체조가 존 재하는 것으로, 자성발생성이배체(gynogenesis diploid) 또는 웅성발생성이배체(androgenesis diploid) 유도 기술에 의해서 생산되어 왔다. 이들은 자외선에 의해서 유전물질이 불활성화 된 정자 또는 난자를 이용하여 유도됨으로써, 오로지 난자 또는 정자의 유전물질만으로 발생하게 된다(Tanaka et al., 2004; Yi et al., 2009). 경골어류의 반수체에 대한 연구는 금붕어(goldfish, Carassius auratus), 송사리(medaka, Oryzias latipes) 및 제브라피 쉬(zebrafish, Danio rerio)에서 보고된 바 있다(Walker, 1999; Araki, 2001; Luo and Li, 2003). 이들의 연구 결과 경골어류 의 반수체는 일반적으로 해로운 열성치사 유전자 존재 또는 유 전자 산물의 부족 때문에 부종(edema), 왜소증(dwarfism), 소 두증(microcephaly) 및 소안구(microphthalmia) 등과 같은 이 형적인 특징이 관찰되었고, 부화 전후 또는 난황 흡수 후 모두 사망하는 것으로 보고되어 왔으며(Felip et al., 1999; Luo and Li, 2003), 이러한 반수체의 특이적인 이상발생 현상은 반수 체 증후군(haploid syndrome)으로 명명되어 왔다(Tvedt et al., 2006). 그러나 반수체는 한 개의 염색체조만을 가짐으로써 우 성 유전자형 또는 열성 유전자형에 대한 표현형을 모두 관찰할 수 있기 때문에 효율적인 유전자 분석 및 검색의 방법론으로 이 용되고 있다(Yi et al., 2009). 이에 제브라피쉬는 반수체 배아의 반수체 넙치(Paralichthys olivaceus)의 발생학적 특성 분석

【Cytogenetic analysis was conducted to obtain basic information for chromosome manipulation of starry flounder Platichthys stellatus. Nuclear surface area and volume of erythrocyte were $7.60{\pm}0.93{\mu}m^2$ and $12.80{\pm}1.75{\mu}m^3$ , respectively. The haploid DNA content of the species was 0.66 pg/haploid cell which correspond to 93% of olive flounder Paralichthys olivaceus. A karyotype analysis was also carried out with the species using conventional staining and Ag-NOR banding techniques. It was consisted of 48 acrocentric chromosomes and inter-sex or intra-individual polymorphism was not detected in all specimens analyzed. The NOR regions, appearing a terminal position of the short arm of the smallest acrocentric pairs.】

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.

Cardiovascular system is the primary organ to develop and reach a functional state, which underscores the essential role of the vasculature in the developing embryo. The vasculature is a highly specialized organ that functions in a number of key physiological works including the carrying of oxygen and nutrients to tissues. It is closely involved in the formation of heart, and hence it is essential for survival during the hatching period. The expression of genes involved during vascular development in the olive flounder (Paralichthys olivaceus) in the days after hatching is not fully understood. Therefore, we examined the expression patterns of genes activated during the development of flounder. Microscopic observations showed that formation of blood vessels is related to the expression of the vimentin gene. Also, the temporal expression patterns of this vimentin-like gene in the developmental stages and in the normal tissues of olive flounder. The purpose of this study was to examine the expression patterns of vimentin in normal tissues of the olive flounder and during the development of the vascular system in newly hatched olive flounders and HIF-1 plays a vital role in the formation of blood vessels during development. Vimentin expression was strong at the beginning of the development of blood vessels, and was present throughout all developmental stages. Our findings have important implications with respect to the roles of vimentin and HIF-1 in the development and evolution of the first blood vessels in olive flounder. Further studies are required to elucidate the vimentin-mediated hypoxic response signal transduction and to decipher the functional role of vimentin in developmental stages.

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.

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.

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.

Division of Animal Science, Gyeongsang National University, 501, Jinjudae-ro, Jinju-si, Gyeongsangnam-do, 660-701, KoreaABSTRACTThis study estimated the genetic parameters of growth-related traits from 11-month-old olive flounder(Paralichthys olivaceus) base population in which wild and cultured broodstocks were introduced. The heritability of body weight, total length, body height, body shape, and condition factor of the base population was estimated to be 0.754, 0.753, 0.789, 0.438, and 0.369, respectively. Genetic correlations between body weight and total length, between body weight and body height, and between total length and body height were 0.969, 0.960, and 0.960, respectively, showing highly positive correlation. Genetic correlations between body weight and body shape, between total length and body shape, between body height and body shape were 0.403, 0.344, and 0593, respectively, showing low negative correlation. Overall, olive flounder might be an appropriate selective breeding species with the high heritability of the traits in an 11-month-old population.(Key words : Olive flounder(Paralichthys olivaceus), Growth-related traits, Genetic parameter, Heritability, 11-month-old)

To estimate the effect of selective breeding on the improvement of growth in olive flounder Paralichthys olivaceus, we compared the growth of a strain selected for rapid growth to that of an unselected strain from a commercial hatchery. The fish strains were fed with either moist pellets (MP) (dry matter, 59.8% crude protein; 14.1% lipid) or extruded pellets (EP) (dry matter, 50.4% crude protein; 13.8% lipid) for 190 days and were reared under similar conditions. The mortality rates were less than 2% and were not significantly different among the experimental groups. The growth rate of the selected fish was significantly greater than that of the unselected fish regardless of the diet type, and both strains fed MP grew significantly faster than those fed EP. The selected fish consumed more feed than the unselected fish. However, there was no significant difference in the feed efficiency between the selected and unselected fish. These results demonstrate that the selected fish exhibited superior growth rates, and that this was associated with a greater intake of food. Thus, selective breeding may be useful for improving the growth of commercial olive flounder.

We have constructed a cDNA library using brain samples of olive flounder Paralichthys olivaceus. Here, we described the study on gene identification by screening 356 clones from the brain cDNA library of olive flounder. Here, we screened 356 clones from the library to identify genes. Of these, 176 (49.5%) were identified as orthologs of known genes from olive flounder and other organisms. Among the 176 EST clones, 33 (18.7%) represented 11 unique genes that are identical to expressed sequence tags (ESTs) reported for olive flounder, and 120 (68.2%) represented 102 unique genes known from other organisms. The percentage of unknown genes (50.5%) is higher than in other olive flounder cDNA libraries (Lee et al., 2003, 2006, 2007), reflecting the high complexity of brain tissue. Further studies of expression characterization and developmental behavior related to these genes should provide useful insight into the physiological functions of the brain in olive flounder.

The aim of this study was to assess whether size-rank at the juvenile stage is associated with growth performance and sex ratio in olive flounder (Paralichthys olivaceus). Juveniles (mean body weight, ) were selected from two age groups, with the high size-rank group (HSRG) fish in the upper 19.3-67.3% of 104 days post-hatching (dph) age group and the low size-rank group (LSRG) fish in the lower 9.0-55.7% of 116 dph age group. Triplicate groups of 100 fish were reared over 32 weeks. A heavier final body weight was observed in the HSRG () compared to the LSRG (, P

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.

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 1,355±742 g of body weight, 48.01±7.79 cm of total length, and 40.96±6.80 cm of body length. Also body height, body shape index and condition factor were 17.19±3.43 cm, 9.99±0.74 and 11.16±1.54, 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

The population structure of olive flounder Paralichthys olivaceus was estimated using nine polymorphic microsatellite (MS) loci in 459 individuals collected from eight populations, including five wild and three hatchery populations in Korea. Genetic variation in hatchery (mean number of alleles per locus, A = 10.2-12.1; allelic richness, A(R) = 9.3-10.1; observed heterozygosity, H(O) = 0.766-0.805) and wild (mean number of alleles per locus, A = 11.8-19.6; allelic richness, A(R) = 10.9-16.1; observed heterozygosity, H(O) = 0.820-0.888) samples did not differ significantly, suggesting a sufficient level of genetic variation in these well-managed hatchery populations, which have not lost a substantial amount of genetic diversity. Neighbour-joining tree and principal component analyses showed that genetic separation between eastern and pooled western and southern wild populations in Korea was probably influenced by restricted gene flow between regional populations due to the barrier effects of sea currents. The pooled western and southern populations are genetically close, perhaps because larval dispersal may depend on warm currents. One wild population (sample from Wando) was genetically divergent from the main distribution, but it was genetically close to hatchery populations, indicating that the genetic composition of the studied populations may be affected by hydrographic conditions and the release of fish stocks. The estimated genetic population structure and potential applications of MS markers may aid in the proper management of P. olivaceus populations.