Functional relation of agouti signaling proteins (ASIPs) to pigmentation and color change in the starry flounder, Platichthys stellatus.

We investigated the involvement of agouti-signaling proteins (ASIPs) in morphological pigmentation and physiological color change in flatfishes. We isolated ASIP1 and 2 mRNAs from the skin of starry flounder (Platichthys stellatus), and compared their amino acid (aa) structures to those of other animals. Then, we examined the mRNA expression levels of two ASIPs (Sf-ASIPs) in the pigmented ocular body and in the unpigmented blind body, as well as in the ordinary skin and in albino skin, in flatfishes. To investigate the role of Sf-ASIPs in physiological color change (color camouflage), we compared the expression of the two genes in two background colors (dark-green and white). Sf-ASIP1 cDNA had a 375-bp open reading frame (ORF) that encoded a protein consisting of 125 aa residues, and Sf-ASIP2 cDNA had a 402-bp ORF that encoded a protein consisting of 132 aa residues. RT-PCR revealed that the strongest Sf-ASIP1 and Sf-ASIP2 expression levels were observed in the eye and blind-skin, respectively. In Sf-ASIP1, the gene expression did not differ between the ocular-side skin and blind-side skin, nor between ordinary skin and abnormal skin of the fish. However, in Sf-ASIP2, the expression level was significantly higher in blind-side skin, compared to ocular-side skin, suggesting that the ASIP2 gene is related to the countershading body pigment pattern of the fish. In addition, the Sf-ASIP2 gene expression level was lower in the pigmented spot regions than in the unpigmented spot regions of the malpigmented pseudo-albino skins on the ocular side, implying that ASIP2 is responsible for the ocular-side pseudo-albino. Additionally, ASIP2 gene expression in the blind-side skin of ordinary fish was enhanced by a white tank, implying that a bright background color could inhibit hypermelanosis in the blind-side skin of cultured flounder by increasing the activity of the Sf-ASIP2 gene. However, we did not find any relationship of ASIPs with camouflage color changes. In conclusion, the ASIP2 gene is related to the morphological pigmentation (countershading and malpigmentation) of the skin in starry flounder, but not with physiological color changes (color camouflage) in the ocular-side skin.

Reproduction and Maturation of Sea Bass, Lateolabrax japonicus, after Transportation from Net-Cages to Indoor Tanks.

To determine whether the reproductive processes of sea bass, Lateolabrax japonicus, proceed normally after transportation from an outdoor net-cage into indoor tanks, we examined changes in the gonadosomatic index (GSI), histological gonadal tissue, and plasma levels of sex hormones (testosterone and estradiol-17ß) during their annual reproductive cycle. We also measured maturation and spawning across two sea water salinity levels (full and low salinity). Fecundity was estimated by the relationship between egg number and body size in female sea bass. Monthly changes in the GSI, histological gonadal tissues, and oocyte size showed both male and female sea bass reach final maturation in January and February, respectively, indicating that the spermiation of males occurs earlier than the spawning of females. The histological results indicated that the sea bass is a multiple spawner, similar to many marine teleosts, exhibiting group-synchronous oocyte development. Female maturation and spawning were enhanced in lower salinity seawater (29.6-31.0 psu) compared to that of normal salinity (34.5-35.1 psu). These results confirm that sea bass reproduction can occur successfully in captivity and imply that fertilized eggs can be collected from February to March. Additionally, our results show that lower salinity enhances oocyte maturation and spawning of female sea bass.

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-ß-LPH, ß-MSH and ß-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 ß-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.

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.

Influence of density and background color to stress response, appetite, growth, and blind-side hypermelanosis of flounder, Paralichthys olivaceus.

To study the relevance of density and background color to stress response, appetite, and growth in olive flounder, Paralichthys olivaceus, we reared two duplicate groups of juveniles (total length 4.46 ± 0.06 cm, body weight 0.77 ± 0.03 g) in flat-bottom aquaria with dark-green (control) and white backgrounds for 120 days. We measured cortisol and glucose levels in blood and calculated the daily food intake, food conversion efficiency, survival rate, and growth rate. To study the relevance of density and background color to malpigmentation (hypermelanosis) on the blind side, we also compared malpigmented ratios and prepro-melanin-concentrating hormone mRNA activities in the brain between the dark-green and white background groups, as well as between a relatively lower density (60 days) and higher density (120 days). Although we measured relatively higher levels of cortisol and glucose in the white background group and over 200 % of coverage area [PCA]), the bright background failed to induce an acute stress response of more than 20 ng/ml cortisol and 40 mg/dl glucose both in 60 days and 120 days, but did enhance appetite and growth. Also, a bright background color delayed hyperpigmentation only at a low density below 200 % PCA, but did not inhibit malpigmentation at a high density of more than 200 % PCA. In addition, below 200 % PCA, expression of MCH mRNA was significantly higher in the white group, but the level was reversed and was lower in the white group at more than 200 % PCA. In conclusion, although did not induce a high stress response over 200 % PCA, the bright background color resulted in a moderate increasing of cortisol level in blood below 20 ng/ml and enhanced appetite and growth. Moreover, at a density below 200 % PCA, the bright color inhibited hypermelanosis with high MCH mRNA activity, but at more than 200 % PCA did not inhibit malpigmentation, and the fish showed low MCH mRNA activity, indicating that the inhibitory effect of a bright background color on hypermelanosis is density dependent.

Effects of gonadotropin-releasing hormone analog (GnRHa) on steroidogenic factor-1 (SF-1) and estrogen receptor beta (ERbeta) gene expression in the black porgy (Acanthopagrus schlegeli).

We examined effects of GnRHa on expression of steroidogenic factor-1 (SF-1) and estrogen receptor beta (ERbeta) in the pituitary and gonad of protandrous black porgy (Acanthopagrus schlegeli). Fish were intraperitoneally injected with 0.2 microg GnRHa/g fish and then pituitary, gonad and plasma were sampled at 0, 6, 12, 24 and 48 h after injection. In gonad, the mRNA levels of the SF-1 were high at 6 h post injection, and then continuously decreased until 24 h; high expression of ERbeta mRNA levels was only observed at 12 h. In contrast, pituitary SF-1 mRNA levels were very low during the experimental period. GnRHa stimulation caused a significant increase of plasma testosterone (T) and estradiol-17beta (E(2)) after 24 h. We suggest that SF-1 and ERbeta play an important role in the development of gonad and these genes are involved with sex change in fish.