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We have previously shown that the somatic growth of barfin flounder, Verasper moseri, was promoted by green light. The present study was undertaken to elucidate whether growth-promoting effect of green light can be observed in other flatfishes and to understand the roles of endocrine systems in green light-induced growth. Herein, we demonstrated facilitation of growth by green light in the spotted halibut, Verasper variegatus, and Japanese flounder, Paralichthys olivaceus. Blue and blue-green light showed potencies that were similar to that of green light, while the potencies of red and white light were equivalent to that of ambient light (control). We also examined the effects of green light on growth and endocrine systems of V. variegatus at various water temperatures. Growth of the fish was facilitated by green light at four different water temperatures examined; the fish were reared for 31 days at 12 and 21 °C, and 30 days at 15 and 18 °C. Increase in condition factor was observed at 15 and 18 °C. Among the genes encoding hypothalamic hormones, expression levels of melanin-concentrating hormone 1 (mch1) were enhanced by green light at the four water temperatures. Expression levels of other genes including mch2 increased at certain water temperatures. No difference was observed in the expression levels of pituitary hormone genes, including those of growth hormone and members of proopiomelanocortin family, and in plasma levels of members of the insulin family. The results suggest that green light may generally stimulate growth of flatfishes. Moreover, it is conceivable that MCH, production of which is stimulated by green light, is a key hormone; it augments food intake, which is intimately coupled with somatic growth., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We previously reported that the somatic growth of barfin flounder, Verasper moseri, was effectively stimulated by the green light compared to the blue and red lights. Herein, we report the effects of different green light intensities on the growth and endocrine system of the fish. Fish were reared in a dark room with light from a light-emitting diode (LED) at a peak wavelength of 518nm under controlled photoperiod (10.5:13.5h, light:dark cycle; 06:00-16:30, light) with three levels of photon flux density (PFD)-2 (low), 7 (medium), or 21 (high) μmol·m -2 ·s -1 at the water surface. The average water temperature was 10.2°C, and the fish were fed until satiety. The fish reared under high PFD of green light showed the highest specific growth rates, followed by the medium PFD group. Under high PFD, the fish showed the highest amount of melanin-concentrating hormone mRNA in their brains and insulin in plasma, while the lowest amount of growth hormone was observed in their pituitary glands. These results suggest that the green light stimulated the growth of barfin flounders in a light intensity-dependent manner in association with their central and peripheral endocrine systems. However, when the fish were reared in an ordinary room where they received both ambient and green LED lights, the fish under LED and ambient light grew faster than those under ambient light only (control). Moreover, no difference was observed in the specific growth rate of the fish reared under the three different green LED light intensities, suggesting that the growth was equally stimulated by the green light within a certain range of intensities under ambient light., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We investigated the effects of specific wavelengths of light on the growth of barfin flounder. The fish, reared in white tanks in a dark room, were irradiated with light from light-emitting diodes (LEDs) with peak wavelengths of 464nm (blue), 518nm (green), and 635nm (red) under a controlled photoperiod (10.5:13.5, light-dark cycle; 06:00-16:30, light). Fish were reared for four weeks in three independent experiments at three different water temperatures (averages of 14.9°C, 8.6°C, and 6.6°C). The fish irradiated with blue and green light had higher specific growth rates (% body weight⋅day(-1)) than fish irradiated with red light. Notably, green light had the greatest effect on growth among the three light wavelengths at 6.6°C. In the brains of fish reared at 6.6°C, the amounts of melanin-concentrating hormone 1 mRNA under green light were lower than those under red light, and amounts of proopiomelanocortin-C mRNA under blue and green light were higher than those under red light. No differences were observed for other neuropeptides tested. In the pituitary, no difference was observed in growth hormone mRNA content. In plasma, higher levels of insulin and insulin-like growth factor-I were observed in fish under green light than those of fish under red light. These results suggest that the endocrine systems of barfin flounder are modulated by a specific wavelength of light that stimulates somatic growth., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Green light irradiation facilitates the somatic growth of barfin flounder (Verasper moseri). However, the V. moseri visual system, which may be associated with somatic growth by acting on the endocrine system upon exposure to this particular wavelength, remains largely unexplored. Herein, we characterized the visual opsin repertoire of V. moseri to understand the molecular basis underlying this effect. The five types of visual opsins that are found in vertebrates were cloned from RNA that was extracted from the eyes of V. moseri. Notably, V. moseri possessed one pseudogene (RH2-A) and two intact (RH2-B and RH2-C) copies of "green-sensitive" opsin genes. The wavelengths of maximum absorption spectra (λmax) for each of the reconstituted photopigments were 552nm for "red-sensitive" LWS, 506nm for RH2-B, 490nm for RH2-C, 482nm and 416nm for "blue-sensitive" SWS2A and SWS2B, respectively, 367nm for "ultraviolet-sensitive" SWS1, and 494nm for "dim-light sensitive rhodopsin" RH1. The λmax of SWS2A was longer than that of any other reported vertebrate SWS2 opsin. By measuring the expression level of these opsin genes with quantitative RT-PCR in 3-, 15-, and 27-month-old fish, we found that RH2-B and SWS2A were expressed at a constant level, whereas the expression of LWS, RH2-C, SWS2B, and SWS1 opsin genes decreased, and that of RH1 increased with age. Barfin flounders inhabit inshore waters at a young age and expand their habitat to deep sea areas as they age, and green light is relatively abundant in deep water compared to the lights of other wavelengths in shallow water. Our results indicate that gene repertoire and expression profile of the opsin genes of barfin flounder are adaptive to their habitat shift that occurs during development, with some opsins acquiring a distinct λmax., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Larval growth and development of hybrid flounder were observed and compared with those of their parent species. The reciprocal hybrids of female starry flounder Platichthys stellatus and male stone flounder Kareius bicoloratus (hybrid Sb) and of female K. bicoloratus and male P. stellatus (hybrid Bs) both survived and grew to juveniles. Development was divided into nine stages (A-I). Many of the hybrids' traits were identical and intermediate to those of their parents. The position of the eye, however, was primarily sinistral in both hybrids (80% in Sb and 76% in Bs), a trait possessed by P. stellatus (80%) in the western Pacific Ocean. The daily growth rates of the larvae were similar. In the parent species, development was more rapid in P. stellatus than in K. bicoloratus whereas rate of development was intermediate in both Sb and Bs hybrids. The size at settlement [standard length (LS ) at stage H (mean ± s.d.)] was 9·82 ± 1·47 mm for the hybrid Sb and 9·99 ± 0·90 mm for the hybrid Bs, while the minimum age at metamorphosis (initial age at stage H) was 29 days after hatching (DAH) in both hybrids. In comparison, LS at settlement in parent species was 6·43 ± 0·25 mm for P. stellatus and 12·87 ± 1·29 mm for K. bicoloratus. Minimum age at metamorphosis for the parents was 23 DAH at stage G in P. stellatus and 34 DAH at stage H in K. bicoloratus. Thus, the timing of settlement of hybrids was different from that of their parent species. These traits may occur with high frequency in a natural habitat., (© 2014 The Fisheries Society of the British Isles.)

The insulinotropic effects of eighteen L-amino acids, two D-amino acids, and glucose were investigated to evaluate the priority of those as stimulators of insulin secretion in barfin flounder (Verasper moseri). This is also the first step in characterizing the insulinotropin-sensing molecule. After intramuscular injection of amino acids or glucose at doses of 3.50 and 1.75 mmol/kg body weight, plasma was collected periodically to determine plasma insulin level. Twelve amino acids and glucose showed insulinotropic effects. Four L-amino acids (Arg, Ala, Met, Ser) produced significantly higher integrated levels of plasma insulin (12.4-34.8 ng/ml) than glucose (average: 4.7 ng/ml) during 3h after injection. D-Amino acids (Arg, Ala) showed no activity. This indicates that many amino acids have strong insulinotropic activities and supports a classic idea, which is well known but has not been confirmed, that amino acids rather than glucose are the important insulinotropins in fish. This study also indicates that the insulinotropic activity of amino acids is restricted to L-amino acids and establishes which amino acids are the strongest stimulators of the insulinotropin sensor in barfin flounder. Co-injection of insulin and L-Thr, L-Ala, or glucose produced a hypoglycemic and hypoaminoacidemic state, indicating that insulin can lower blood amino acid level as well as blood sugar level. This study suggests that insulin plays a more important role than glucose in the regulation of blood L-amino acid metabolism, at least in flounder.

A new method for visualizing small and multiple phosvitins (Pvs) in oocytes from a marine teleost was developed by a combination of gel filtration, alkaline phosphatase treatment, and SDS-PAGE followed by silver staining. Three distinct Pv polypeptides having molecular masses of 15 kDa, 8 kDa, and 7 kDa were visualized in vitellogenic follicle extract of barfin flounder, Verasper moseri. N-terminal amino acid sequencing identified two different N-termini that fell into the PvA (7 kDa) and PvB (15 kDa and 8 kDa) groups, which were derived from two forms of vitellogenin (Vg), VgA and VgB, respectively. Analysis of time-course change in phosphorus-rich peaks of gel chromatography fractions of follicle extracts from different maturational stages demonstrated a rapid degradation of Pvs during mid-phase of oocyte maturation. Quantitative analysis of free amino acids in maturing follicles revealed an increment of serine content but not of phosphoserine, indicating the occurrence of dephosphorylation concomitant with Pv degradation. Measurement of phosphatase activity in follicles and eggs at different maturational stages demonstrated a significant activation of phosphatase especially under acidic conditions. This suggested that Pv degradation and dephosphorylation are regulated by changes in ooplasm pH during oocyte maturation. Our results also suggested that the Pvs in barfin flounder vitellogenic oocytes bind to much lower amounts of calcium and magnesium than those of masu salmon, Oncorhynchus masou. This indicates that the Pvs in the barfin flounder, a marine teleost spawning its eggs in seawater, do not play a role in the transport and deposition of calcium and magnesium into oocytes.

A time-resolved fluoroimmunoassay system (TR-FIA) for measuring flounder insulin-like growth factor-I (IGF-I) was developed using biotinylated flounder IGF-I, anti-fish IGF-I antiserum and europium-avidin conjugate. The detection limit per well was <5 pg/well corresponding to <0.5 ng/ml in a basic procedure for sample of 10 microl/well and to <0.08 ng/ml in a procedure modified for high volume samples (up to 70 microl/well). Specificity of the assay was validated using various IGF-Is and insulins. All IGFs except seabream IGF-I showed very low or no crossreactivity. Binding inhibition curves for flounder and seabream IGF-Is were completely identical to each other. Intra- and interassay variations ranged from coefficients of variations of 3.9% to 7.2%. Recovery tests using barfin flounder plasma varied from 82.7 to 101.6% in the added range from 20 to 160 ng/ml. This assay system was applied for measuring total plasma IGF-I in barfin flounder injected porcine growth hormone (GH). A group injected with GH at the dose of 0.05 IU/gBW showed a significant increase of total plasma IGF-I compared with those of albumin-injected (control) and initial groups. In addition, I was able to substitute time-resolved fluorometric detection in this assay system with enzymatic fluorometric detection (FIA). Binding inhibition curve for flounder IGF-I in this substituted assay system showed equal performance with that of the TR-FIA system. Correlation of IGF-I levels between TR-FIA and FIA was high (r(2)=0.957) in plasma samples from barfin flounders in various physiological conditions. Thus, the present study shows precision and efficiency of two non-radioisotopic immunoassay systems for measuring flounder IGF-I.

The concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), non-ortho polychlorinated biphenyls (non-ortho PCBs), and mono-ortho polychlorinated biphenyls (mono-ortho PCBs) in Japanese flounder (Paralichthys olivaceus, body length 10.4-36.6 cm) collected from Sendai Bay, Japan, were determined using high-resolution gas chromatography-mass spectrometry. The relationship between the concentrations of these compounds (dioxins) and the body length of the Japanese flounder was examined. The total PCDD and total PCDF concentrations did not correlate with body length (both r(2) < 0.1, both p > 0.05), whereas the total non-ortho PCB and total mono-ortho PCB (coplanar polychlorinated biphenyls, Co-PCBs) concentrations were significantly correlated (r(2)= 0.8, p < 0.05 and r(2)= 0.63, p < 0.05, respectively). The bioaccumulation properties of PCDD/Fs in Japanese flounder differed from those of Co-PCBs. Toxicity equivalency quotient (TEQ) values derived from the Co-PCBs made up 46.3%-63.7% of the total TEQ value for all the dioxins. Although the concentrations of non-ortho PCBs were lower than those of mono-ortho PCBs, the TEQ value for non-ortho PCBs was higher than that for mono-ortho PCBs. The TEQ value for non-ortho PCBs increased more with increasing body length than did the values for PCDDs, PCDFs, and mono-ortho PCBs. These results show that from the standpoint of risk management, non-ortho Co-PCBs are the most important of the dioxins in Japanese flounder.

A time-resolved fluoroimmunoassay (TR-FIA) system was developed to quantify insulin levels in the barfin flounder. This TR-FIA system is a solid-phase assay based on competition of unlabeled insulins and biotinylated barfin flounder insulin-II against an anti-barfin flounder insulin-II antibody. The minimum detectable level of barfin flounder insulin-I and -II in this TR-FIA was 10 pg/well which corresponded to 1.0 ng/ml, and insulin-II showed slightly higher crossreactivity than insulin-I. The accuracy of this TR-FIA was assured by specificity test, validation test, and recovery test using plasma added insulin-II. The results indicated the high specificity and sufficient accuracy of this assay system for insulin level measurement. This system was applied to the measurement of plasma insulin levels of fed and fasted barfin flounders. Plasma insulin levels (average +/- SEM) in fed flounders reached a maximum 2 h (9.3 +/- 1.7 ng/ml) and decreased gradually thereafter, while those in fasted flounders remained at low levels (1.1 +/- 0.1-2.0 +/- 0.2 ng/ml) during the experiment. After removing proteins by acidification and subsequent gel filtration, plasma samples taken from fed and fasted flounders at 2 h after feeding were fractionated separately by reversed-phase HPLC. In fed flounders, insulin immunoreactivity was detected in fractions corresponding to those of insulin-I or -II. The ratio of integrated insulin immunoreactivities of each peak was 0.378 +/- 0.044 (average +/- SD). This value was in good agreement with those (0.355 +/- 0.019) of absorbance areas of each insulin from Brockmann body extracts of the barfin flounder on reversed-phase HPLC. In fasted flounders, very weak insulin immunoreactivities were observed at retention times corresponding to those of insulin-I and -II. These results indicated that both insulin-I and -II were secreted into the blood being induced by feeding stimulation with approximately the same ratio as that of the quantities harbored in the Brockmann body., ((C)2002 Elsevier Science (USA).)

We cloned and sequenced the complete mitochondrial genome of Japanese flounder (Paralichthys olivaceus). A circular 17,090 bp mitochondrial genome from the flounder contains 37 structural genes as in other vertebrates so far reported. This is the first report of the complete mitochondrial sequence from a higher teleostean fish (Acanthopterygii). The organization including gene order is quite similar to that of other teleostean fishes as well as placental mammals. The putative control region of the Japanese flounder mitochondrial genome contains a length variable region of about a 74 bp tandem repeat cluster. As a preliminary study we adopted the maximum likelihood and neighbor-joining inference methods to examine phylogenetic relationships among teleostean and related fishes. Comparisons of amino acid sequences of protein-coding genes and nucleotide sequences of tRNA genes resolved some middle to deep branches among some teleostean fishes. The flounder mitochondrial genome does not show an indication of evolutionary rate difference among teleosts leading to difficulty in phylogenetic analyses, and our data is useful for future evolutionary studies dealing with higher teleostean fishes.

Two forms of vitellogenin (Vg), Vg A and Vg B, were identified in serum from estrogen-treated barfin flounder (Verasper moseri). Structural changes of lipovitellins (Lvs) derived from the two Vgs were examined during vitellogenesis and oocyte maturation. Two Lvs, vLv A and vLv B, were identified electrophoretically and immunologically in postvitellogenic oocytes. Each appeared to be composed of distinct heavy chains (vLvH A, M(r) 107,000, and vLvH B, M(r) 94,000) and light chains (vLvL A, M(r) 30,000, and vLvL B, M(r) 28,000) when analyzed by SDS-PAGE. Results from N-terminal amino acid sequencing and Western blotting using antisera to vLvH A and vLvH B verified that there are two Vg polypeptides in serum from estrogen-treated fish, Vg A (M(r) 168,000) and Vg B (M(r) 175,000), which give rise to vLvH A-vLvL A and vLvH B-vLvL B, respectively. N-terminal sequencing revealed two sequences for both phosvitin and beta'-component, supporting the concept of duality for all three classes of Vg-derived yolk proteins. During oocyte maturation, native dimeric vLv B was dissociated into a native M(r) 170,000 monomer (oLv B). Meanwhile, vLv A was extensively cleaved including complete degradation of vLvH A into free amino acids. We propose that the quantitative ratio of vLv A to vLv B in postvitellogenic oocytes regulates the buoyancy of the spawned pelagic eggs by controlling availability of free amino acids which function as osmotic effectors during oocyte hydration. The vLv A/vLv B ratio likely also controls the proportional availability of different types of nutrients, free amino acids versus Lv, for use during embryonic development., (Copyright 1999 Academic Press.)