Duk-Young Kang

Functional characterization of two melanin-concentrating hormone genes in the color camouflage, hypermelanosis, and appetite of starry flounder.

To investigate the involvement of two melanin-concentrating hormones (MCHs) in skin color change and appetite in flatfish, we isolated two forms of prepro-melanin concentrating hormone (pMCHs) mRNA in the starry flounder Platichthys stellatus and compared their amino acid structures to those of other animals. Then, we examined the relationship of the two starry flounder pMCH (sf-pMCH) with physiological color change, blind-side malpigmentation, and feeding by quantifying mRNA expression level. Sf-pMCH1 cDNA had a 387-bp open reading frame (ORF) that encoded a protein consisting of 129 amino acid residues. The sf-pMCH1 protein included a signal peptide composed of 24 amino acid residues; MCH1 encoded a protein consisting of 17 amino acids. The sf-pMCH2 cDNA had a 450-bp ORF that encoded a protein consisting of 150 amino acid residues, which included a signal peptide comprising 23 amino acid residues; MCH2 encoded a protein consisting of 23 amino acids that was structurally similar to mammalian MCH. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that the strongest sf-pMCHs gene expression was observed in the brain and pituitary, but weak or no amplification was detected in other tissues. The expression of sf-pMCH1 was relatively high compared to that of sf-pMCH2 in the brain. The relative levels of mRNA were significantly lower in dark background-reared and hypermelanic fish, indicating that the two pMCHs and background color are related to the physiological and morphological color changes of skin. In term of feeding regulation, we found an obvious functional role of pMCH1 in appetite, whereas the pMCH2 gene was not found to play a role in feeding.

Morphological Specificity in Cultured Starry Flounder Platichthys stellatus Reared in Artificial Facility

The starry flounder Platichthys stellatus, like all flatfish, exhibits conspicuous lateral asymmetry in numerous traits, most obvious of which is the migration of one eye to the other side of the head during metamorphosis. Additional changes related to eye migration include asymmetrical pigmentation, and a behavioral shift from larvae that exhibit upright, open-water swimming to juveniles and adults that lie on the ocean floor, eye side up. However, the morphology of these juveniles has been quite plastic in recent years, a phenomenon which is thought to be related to a diverse suite of semi-intensive and intensive larviculture methods. The cause of morphological abnormalities in the farmed flatfish is poorly understood. In the present study, we observe the features of morphological specificity and abnormality of immature fish (mean total length 23 cm) and survey the occurrence frequency of the specificity and abnormality of juvenile (mean total length 6.70 cm) in artificial culture facility. We find 2 types of abnormality (e.g., albino in ocular side and hypermelanosis in blind side) and 1 type of specificity (e.g., lateral polymorphism). These considerably differ from normal individuals (has sinistral eye and pigmented on only one side) by several characteristics (dextral eye, ocular side albinism, blind side hypermelanosis). The incidences of albinism, hypermelanosis, and body reversal are 10.1 ± 2.56%, 91.7 ± 1.7%, and 13.1 ± 1.1%, respectively. These suggest that these morphometric and morphological differences occur more in artificial environment during and just after metamorphosis.

Effects of Stocking Density on the Blind-side Hypermelanosis of Cultured Olive Flounder Paralichthys olivaceus

non-eyed (blind) side in duplicate at densities of 150 individuals/m 2 (commercial production density: control) and 450 individuals/m 2 (high density group) for 90 days in 1-t dark-green fiberglass reinforced plastics (FRP) tank. We recorded feed intake, feed conversion efficiency (FE), growth and survival, and measured the ratios of staining blind-side area (staining area) and ambicolored fish every 30 days. Daily feed intake (DFI), feed conversion efficiency (FE), growth rate, condition factors, and survival rate were calculated at the end of the experiment. Although the FE was higher in the high-density compared to the control, the two density groups showed similar feed intakes, growth, and survival. The ratio of staining area as well as the ratio of ambicolored fish significantly were significantly higher in the high-density group than in the control from days 30 to 60, but significantly increased and evened out by the end of the experiment (P< 0.05). In conclusion, we determined that rearing density is not the main cause of the blind-side hypermelanosis, but found that increasing the rearing density can accelerate the ambicoloration in olive flounders.

Functional relevance of three proopiomelanocortin (POMC) genes in darkening camouflage, blind-side hypermelanosis, and appetite of Paralichthys olivaceus

To determine whether proopiomelanocortin (POMC) genes are involved in darkening color camouflage, blind-side hypermelanosis, and appetite in flatfish, we isolated and cloned three POMC genes from the pituitary of the olive flounder (Paralichthys olivaceus) and compared their amino acid (aa) structures to those of POMC genes from other animals. Next, we examined the relationship of these pituitary POMC genes to camouflage color change, blind-side hypermelanosis, and appetite by quantifying mRNA expression. Olive flounder (of)-POMC1, 2, and 3 cDNAs consisted of 648-bp, 582-bp, and 693-bp open reading frames (ORF) encoding 216 aa, 194 aa, and 231 aa residues, respectively. Structurally, the three of-POMC cDNAs consisted of seven peptides (signal peptide, N-POMC, α-MSH, CLIP, N-β-LPH, β-MSH and β-END [or END-like peptide]) that are similar to those of other fish POMC cDNAs. α-MSH encoded a protein composed of 13 aa and β-MSH encoded a protein composed of 17 aa. The three POMC genes were predominantly expressed in the pituitary gland, but they were also expressed in a variety of tissues, including brain, eye, kidney, heart, testis, and skin. of-POMC2 exhibited the highest expression, while of-POMC3 displayed the lowest expression. The relative levels of of-POMC1 and 3 mRNAs were not influenced by background color and feeding (or fasting), but the relative level of of-POMC2 mRNA significantly increased in response to a dark background and fasting. The relative levels of of-POMC1 and 2 mRNAs were significantly higher in hypermelanic fish; however, we did not determine a direct anorexigenic or orexigenic relationship for the three POMC genes. These results indicate that pituitary POMC genes are related to darkening color change and the differentiation of pigment cells, but they are not directly related to appetite.

Morphological Analysis of Blind-Side Hypermelanosis of the Starry Flounder, Platichthys stellatus during Early Development

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°C 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.

Effect of the Burrowing Substratum on the Growth and Ambicoloration of Juvenile Flounder Paralichthys olivaceus Cultured at High Density

넙치(Paralichthys olivaceus)는 높은 경제적 가치로 국내 양 식 대상 종 최고 많은 생산량을 자랑하며, 국내 연안자원 조성용 어류로 가장 오랫동안 연안에 방류된 종이다(Seo et al., 2010). 그로 인해 국내 연안에서 포획되는 넙치의 40-80%가 방류산이 며, 천연형질의 자연산 비율은 과거에 비해 매우 감소되고 있다 (Jeong and Jeon, 2008). 이 방류산 넙치는 자연산과 다른 한가 지의 형태적 특성을 지니고 있다. 그것은 눈이 없는 무안측에 비 정상적으로 색소세포가 분화하여 체색반문을 형성한다는 점이 다 (Matsumoto and Seikai, 1992; Seikai, 1992). 이러한 특이 적인 형태 특성은 현재 자연산과 양식산 넙치를 구분하는 중요 한 기준으로 활용되고 있다(Gartner, 1986; Burton and Vokey, 2000; Ivankova and Ivankov, 2006; Ivankov et al., 2008). 그런 데 최근 연구(MFAFF, 2010)를 통해 이 증세가 가족력을 지니 고 있을 가능성이 높을 것으로 가계도 조사를 통해 확인되면서, 이러한 기형적 형태 발생이 유전력에 의한 것일 가능성이 조심 스럽게 제시된다. 따라서 지속적인 흑화 개체의 방류는 자연계 내에서 해당 유전형질 고착화로 인해, 몇 세대 후에는 천연형질 의 자연산 개체들은 사라지고 대부분 기형 형질 개체들로 대처 될 수도 있다. 그러므로 천연 형질의 종묘가 생태계적 측면에서 요구되고 있지만, 체색기형 개체의 생산 예방책이나 제어기술 은 아직 여기에 이르지 못하고 있다. 현재 넙치를 포함한 가자미 목 어류의 무안측 체색발현 원인은 밀도(Takahashi, 1994), 수 조 색상(Yamanome et al., 2005; Yamanome et al., 2007), 영 양원(Haga et al., 2004; Tarui et al., 2006) 등 다양한 사육 환 경인자가 제시되지만, 여전히 어떤 요소가 이 현상을 촉발시키 는 주 요인인지 밝혀져 있지 않다(Venizelos and Benetti, 1999; 넙치(Paralichthys olivaceus) 치어의 성장 및 양면착색 현상에 있어 잠입기질의 효과 강덕영*·김효찬·명정인·민병화

Influences of Temperature and Density on the Feeding Growth, and Blind-side Malpigmentation of Fry Starry Flounder Platichthys stellatus

To examine the influences of water temperature and stocking density on feeding, growth and blind-side hypermelanosis of the starry flounder Platichthys stellatus, we performed an experiment with fry at two densities: 2 and 8 fish/L. The fry had a total length of , body weight , and pigmented patches (pigmented ratio ) on the blind side. Duplicate experiments were conducted in 93.7 L glass tanks for 120 days, from July to November. We determined daily food intake (DFI), food efficiency (FE), growth, survival rate, ratio of hypermelanic fish, and pigmented area rate on the blind side at 60-day intervals. The DFI was less than 50 mg/fish/day, and growth was delayed from July to September (water temperature [WT]> ). After October, when WT , the DFI increased significantly and the growth was accelerated, suggesting that the rearing temperature of starry flounder in artificial facilities should be . While the FE, and survival did no differ between the two density groups, DFI and growth were significantly higher at 2 fish/L than at 8 fish/L. There was no difference in the pigmented area ratio between the two density groups. Although the hypermelanosis was not correlated with body size, the malpigmentation increased with growth. These results suggest that a high stocking density is not the main cause of blind-side hypermelanosis, although it can accelerate hypermelanosis in the starry flounder.

The Functional Relevance of Prepro-melanin Concentrating Hormone (pMCH) to Skin Color Change, Blind-side Malpigmentation and Feeding of Oliver Flounder Paralichthys olivaceus

To assess the functional structure of prepro-melanin-concentrating hormone (pMCH), we isolated and cloned pMCH (of-pMCH) mRNA from the brain of the olive flounder, Paralichthys olivaceus, and compared its amino acid sequence with those from other animals. In addition, to examine whether activation of the brain of-pMCH gene is influenced by background color, density, and feeding, we compared pMCH mRNA activities against different background colors (bright and dark) and at different densities (100% PCA and 200% PCA). To examine whether the pMCH gene is related with malpigmentation of blind-side skin and appetite, we compared pMCH gene expression between ordinary and hypermelanic flounders, and between feeding and fasting flounders. The of-pMCH cDNA was 405 bp in the open reading frame [ORF] and encoded a protein of 135 amino acids; MCH was 51 bp in length and encoded a protein of 17 amino acids. An obvious single band of the expected size was obtained from the brain and pituitary by RT-PCR. In addition, of-pMCH gene activity was significantly higher in the bright background only at low density ( 200% PCA), and in hypermelano fish. These results suggest that skin whitening camouflage of the flounder is induced by high MCH gene activity, and the density disturbs the function of background color in the physiological color change. Moreover, our data suggest that a low level of MCH gene activity may be related to malpigmentation of the blind-side skin. In feeding, although pMCH gene activity was significantly increased by feeding in the white background, the pMCH gene activity in the dark background was not influenced by feeding, indicating that the MCH gene activity increased by feeding can be offset by dark background color, or is unaffected by appetite. In conclusion, this study showed that MCH gene expression is related to ocular-skin whitening camouflage and blind-skin hypermelanosis, and is influenced by background color and density.

Influence of Rearing Environmental Factors on Intra-cohort Cannibalism and Growth of Fry in Cultured Puffer Takifugu obscurus

To investigate whether the rearing environmental factors (size heterogeneity, rearing density, and feeding frequency) affect the growth and cannibalism of fry in cultured puffer Takifugu obscurus, we conducted three experiments. To examine size effects, we used small (total length[TL] 26.0±0.5mm, body weight[BW] 0.18±0.01g), medium (TL 23.1±0.1mm, BW 0.28±0.01g), large (TL 26.0±0.2mm, BW 0.48±0.01g) and small-large size combination groups. For density, we used size-matched puffers (TL 25.0±0.5mm, BW 0.53±0.03g) and five density were examined ranging from 1.43 to 7.14 individuals/L. For the feeding frequency, we also used size-matched fry and feeding frequencies of three times/2days, one time/day, two times/day, three times/day. We ran each experiment in triplicate and investigated the survival rate, daily food intake (DFI), feed efficiency (FE), and daily growth rate (DGR). The growth of the puffers increased with increasing size, density, and feeding frequency, while cannibalism increased with a greater size gap, density and lower feeding frequency. Therefore, we concluded that size, rearing density, and feeding frequency are major factors influencing growth and cannibalism of the puffer, T. obscurus.