Effects of Water Temperature on the Body Color and Expression of the Genes Related to Body Color Regulation in the Goldfish.

Melanin-concentrating hormone (MCH), melanocyte-stimulating hormone (MSH), and somatolactin (SL) in the hypothalamus-pituitary axis are associated with body color regulation in teleost fish. Although these hormones' production and secretion respond well to light environments, such as background color, little is known about the effects of different water temperatures. We investigated the effects of water temperature, 10°C, 20°C, and 30°C, on body color and the expression of these genes and corresponding receptor genes in goldfish. The body color in white background (WBG) becomes paler at the higher water temperature, although no difference was observed in black background (BBG). Brain mRNA contents of proMCH genes (pmch1 and pmch2) increased at 30°C and 20°C compared to 10°C in WBG, respectively. Apparent effects of background color and temperature on the pituitary mRNA contents of a POMC gene (pomc) were not observed. The pituitary mRNA contents of the SLα gene were almost double those on a WBG at any temperature, while those of the SLß gene (slb) at 30°C tended to be higher than those at 10°C and 20°C on WBG and BBG. The scale mRNA contents of the MCH receptor gene (mchr2) in WBG were higher than those in BBG at 30°C. The highest scale mRNA contents of MSH receptor (mc1r and mc5r) on BBG were observed at 20°C, while the lowest respective mRNA levels were observed at 30°C on WBG. These results highlight the importance of temperature for the endocrinological regulation of body color, and darker background color may stabilize those endocrine functions.

Effect of Blue Light on the Growth of the Red Swamp Crayfish Procambraus clarkii Larvae -Seasonal and Sexual Differences.

Organisms have the ability to adapt their behavior and physiology in response to seasonal changes in their habitat's environments. Although it is known that a specific light wavelength affects growth and reproduction in various animal taxa, its effect on sexual and seasonal differences in year-round breeding animals remains unclear. Here, we demonstrate that a blue light stimulus promotes or suppresses larval growth in the red swamp crayfish Procambarus clarkia depending on the season. During the spawning season (natural growing period), blue light irradiation accelerates female growth faster than in males, but suppresses growth in both females and males in the overwintering season. Moreover, these seasonal plastic effects of blue light show apparent sexual differences, with female juveniles exhibiting the greatest sensitivity. Our findings provide an opportunity to research how the red swamp crayfish can adapt to various habitable niches from the point of view of light color perception, and can be applied for the development of a more effective aquaculture system, not only for crayfish, but also for other commercially available decapod crustaceans using a specific light environment.

Taisho-Sanshoku koi have hardly faded skin and show attenuated melanophore sensitivity to adrenaline and melanin-concentrating hormone.

Introduction: Koi carp, an ornamental fish derived from the common carp Cyprinus carpio (CC), is characterized by beautiful skin color patterns. However, the mechanism that gives rise to the characteristic vivid skin coloration of koi carp has not been clarified. The skin coloration of many teleosts changes in response to differences in the background color. This change in skin coloration is caused by diffusion or aggregation of pigment granules in chromatophores and is regulated mainly by sympathetic nerves and hormones. We hypothesized that there would be some abnormality in the mechanism of skin color regulation in koi carp, which impairs skin color fading in response to background color. Methods: We compared the function of melanin-concentrating hormone (MCH), noradrenaline, and adrenaline in CC and Taisho-Sanshoku (TS), a variety of tri-colored koi. Results and Discussion: In CC acclimated to a white background, the skin color became paler and pigment granules aggregated in melanophores in the scales compared to that in black-acclimated CC. There were no clear differences in skin color or pigment granule aggregation in white- or black-acclimated TS. The expression of mch1 mRNA in the brain was higher in the white-acclimated CC than that in the black-acclimated CC. However, the expression of mch1 mRNA in the brain in the TS did not change in response to the background color. Additionally, plasma MCH levels did not differ between white- and black-acclimated fish in either CC or TS. In vitro experiments showed that noradrenaline induced pigment aggregation in scale melanophores in both CC and TS, whereas adrenaline induced pigment aggregation in the CC but not in the TS. In vitro administration of MCH induced pigment granule aggregation in the CC but not in the TS. However, intraperitoneal injection of MCH resulted in pigment granule aggregation in both CC and TS. Collectively, these results suggest that the weak sensitivity of scale melanophores to MCH and adrenaline might be responsible for the lack of skin color change in response to background color in the TS.

Effects of background color and feeding status on the expression of genes associated with body color regulation in the goldfish Carassius auratus.

Alpha-melanocyte-stimulating hormone (α-MSH), a peptide derived from proopiomelanocortin (POMC), and melanin-concentrating hormone (MCH), act as neuromodulators and regulate food intake in vertebrates. In teleosts, these peptides are also involved competitively in body color regulation; α-MSH induces a dark body color, while MCH induces a pale body color. Similarly, members of the growth hormone (GH) family, somatolactin (SL) and prolactin (PRL), which are involved in the regulation of energy metabolism, are also associated with body color regulation in teleosts. Since these hormones are involved in both body color regulation and energy metabolism, it is possible that feeding status can affect body color. Here, we examined the effects of fasting on the response of goldfish body coloration to changes in background color. Goldfish were acclimated for one week in tanks with a white or black background under conditions of periodic feeding or fasting. The results showed that body color and expression levels of pmch1 and pomc were affected by background color, irrespective of feeding status. Expression levels of sla were higher in fish maintained in tanks with a black background than in tanks with a white background, and higher in the fasted fish compared to the fed fish. However, the pattern of slb expression was almost the opposite of that observed in sla expression. The expression levels of gh and prl in the pituitary, and pmch2a and pmch2b in the brain, were not affected by background color. These results suggest that MCH, α-MSH, SLα, and SLß might be involved in body color regulation and that they are affected by background color in goldfish. The results also suggest that feeding status may affect body color regulation via SLα and SLß, although these effects might be limited compared to the effect of background color.

Expression dynamics of visual opsin genes in marbled sole Pseudopleuronectes yokohamae during metamorphosis from the larval to the juvenile stage.

We clarified the properties of visual opsin genes in the marbled sole (Pseudopleuronectes yokohamae) by cDNA sequencing, quantification of the opsin gene expression from the larval to the juvenile stage, and measurement of the maximum absorption spectra (λmax) using photopigment reconstitution. In the marbled sole eye, at least eight visual opsin genes, lws, rh2-a, rh2-b, rh2-c, sws2a, sws2b, sws1, and rh1, were expressed. Quantitative RT-PCR analysis revealed that the expression of opsin genes increased (lws, rh2-c, sws2a, and rh1) or decreased (rh2-a, rh2-b, sws2b, and sws1) from the larval to the juvenile stage. Notably, rh2-a expression was observed only in pre- to mid-metamorphic stage larvae and disappeared after metamorphosis. Thus, pre-metamorphism-specific expression of rh2-a in the marbled sole suggests that its function is restricted to the developmental stage. The reconstituted RH2-A opsin λmax was 470 nm, which is typical of acanthopterygian species. These results strongly suggest that mid-wavelength-sensitive rh2-a expression was diminished drastically in the marbled sole, probably resulting in a shift of spectral sensitivity during its metamorphosis from the larval to the juvenile stage.

Effects of tank color brightness on the body color, somatic growth, and endocrine systems of rainbow trout Oncorhynchus mykiss.

We investigated the effects of tank brightness on body color, growth, and endocrine systems of rainbow trout (Oncorhynchus mykiss). Five different tank colors that produce varying levels of brightness were used, including black, dark gray [DG], light gray [LG], white, and blue. The fish were reared in these tanks for 59 days under natural photoperiod and water temperature. The body color was affected by tank brightness, such that body color brightness was correlated with tank brightness (white-housed ≥ LG-housed ≥ DG-housed ≥ blue-housed ≥ black-housed). No difference in somatic growth was observed among the fish reared in the five tanks. The mRNA levels of melanin-concentrating hormone (mch1) was higher in white-housed fish than those in the other tanks, and the mRNA levels of proopiomelanocortins (pomc-a and pomc-b) were higher in fish housed in a black tank than those in other tanks. mRNA level of somatolactin, a member of growth hormone family, was higher in black-housed fish than those in white-housed fish. The mRNA levels of mch1 and mch2 in blue-housed fish were similar to those in black-housed fish, while the mRNA levels of pomc-a and pomc-b in blue-housed fish were similar to those in white-housed fish. The current results suggest that tank color is not related to fish growth, therefore any color of conventional rearing tank can be used to grow fish. Moreover, the association between somatolactin with body color changes is suggested in addition to the role of classical MCH and melanophore stimulating hormone derived from POMC.

The effects of chromatic lights on body color and gene expressions of melanin-concentrating hormone and proopiomelanocortin in goldfish (Carassius auratus).

In the present study, the effects of photic environments, such as background color (white and black) and chromatic lights (blue, green, and red), on body color and gene expressions of melanin-concentrating hormone (mch) in the brain and proopiomelanocortin (pomc) in the pituitary, as well as the roles of the eyes and brain as mediators of ambient light to these genes, were examined in goldfish (Carassius auratus). Body color of goldfish exposed to fluorescent light (FL) under white background (WBG) was paler than those under black background (BBG). Gene expression levels for mch and pomc were reciprocally different depending on background color; under WBG, mRNA levels of mch and pomc were high and low, respectively, while under BBG, these levels were reversed. mch and pomc mRNA expressions of the fish exposed to chromatic light from LED were primarily similar to those exposed to FL, while blue light stimulated the expressions of mch and pomc. Ophthalmectomized goldfish exposed to FL or blue light showed minimum expression levels of mch gene, suggesting that eyes are the major mediator of ambient light for mch gene expression. Contrastingly, mRNA expressions of pomc in ophthalmectomized goldfish exposed to FL were different from those of intact goldfish. These results suggest that eyes play a functional role in mediating ambient light to regulate pomc gene expression. Since ophthalmectomy caused an increase in pomc mRNA contents in the fish exposed to blue light, we suggest that the brain is an additional mediator to regulate pomc gene expression.

Effects of green light on the growth of spotted halibut, Verasper variegatus, and Japanese flounder, Paralichthys olivaceus, and on the endocrine system of spotted halibut at different water temperatures.

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.

Melanin-concentrating hormone is a major substance mediating light wavelength-dependent skin color change in larval zebrafish.

Melanosome dispersion is important for protecting the internal organs of fish against ultraviolet light, especially in transparent larvae with underdeveloped skin. Melanosome dispersion leads to dark skin color in dim light. Melanosome aggregation, on the other hand, leads to pale skin color in bright light. Both of these mechanisms are therefore useful for camouflage. In this study, we investigated a hormone thought to be responsible for the light wavelength-dependent response of melanophores in zebrafish larvae. We irradiated larvae using light-emitting diode (LED) lights with peak wavelengths (λmax) of 355, 400, 476, 530, and 590 nm or fluorescent light (FL) 1-4 days post fertilization (dpf). Melanosomes in skin melanophores were more dispersed under short wavelength light (λmax ≤ 400 nm) than under FL. Conversely, melanosomes were more aggregated under mid-long wavelength light (λmax ≥ 476 nm) than under FL. In addition, long-term (1-12 dpf) irradiation of 400 nm light increased melanophores in the skin, whereas that of 530 nm light decreased them. In teleosts, melanin-concentrating hormone (MCH) aggregates melanosomes within chromatophores, whereas melanocyte-stimulating hormone, derived from proopiomelanocortin (POMC), disperses melanosomes. The expression of a gene for MCH was down-regulated by short wavelength light but up-regulated by mid-long wavelength light, whereas a gene for POMC was up-regulated under short wavelength light. Melanosomes in larvae (4 dpf) exposed to a black background aggregated when immersing the larvae in MCH solution. Yohimbine, an α2-adrenergic receptor antagonist, attenuated adrenaline-dependent aggregation in larvae exposed to a black background but did not induce melanosome dispersion in larvae exposed to a white background. These results suggest that MCH plays a key role in the light wavelength-dependent response of melanophores, flexibly mediating the transmission of light wavelength information between photoreceptors and melanophores.

α-Melanocyte-stimulating hormone promotes bone resorption resulting from increased osteoblastic and osteoclastic activities in goldfish.

We examined the effects of α-melanocyte-stimulating hormone (α-MSH) on bone metabolism using regenerating goldfish scales. Normally developed scales on the bodies of goldfish were removed to allow the regeneration of scales under anesthesia. Thereafter, the influence of α-MSH on the regeneration of goldfish scales was investigated in vivo. In brief, α-MSH was injected at a low dose (0.1 µg/g body weight) or a high dose (1 µg/g body weight) into goldfish every other day. Ten days after removing the scales, we collected regenerating scales and analyzed osteoblastic and osteoclastic activities as respective marker enzyme (alkaline phosphatase for osteoblasts, tartrate-resistant acid phosphatase for osteoclasts) activity in the regenerating scales as well as plasma calcium levels. At both doses, osteoblastic and osteoclastic activities in the regenerating scales increased significantly. Plasma calcium concentrations in the α-MSH-treated group (high doses) were significantly higher than those in the control group. Next, in vitro experiments were performed to confirm the results of in vivo experiments. In the cultured regenerating scales, osteoblastic and osteoclastic activities significantly increased with α-MSH (10-7 and 10-6 M) treatment. In addition, real-time PCR analysis indicated that osteoclastogenesis in α-MSH-treated scales was induced by the receptor activator of the NF-κB/receptor activator of the NF-κB ligand/osteoprotegerin pathway. Furthermore, we found that α-MSH receptors (melanocortin receptors 4 and 5) were detected in the regenerating scales. Thus, in teleosts, we are the first to demonstrate that α-MSH functions in bone metabolism and promotes bone resorption via melatonin receptors 4 and/or 5.

Green-shifting of SWS2A opsin sensitivity and loss of function of RH2-A opsin in flounders, genus Verasper.

We identified visual opsin genes for three flounder species, including the spotted halibut (Verasper variegatus), slime flounder (Microstomus achne), and Japanese flounder (Paralichthys olivaceus). Structure and function of opsins for the three species were characterized together with those of the barfin flounder (V. moseri) that we previously reported. All four flounder species possessed five basic opsin genes, including lws, sws1, sws2, rh1, and rh2. Specific features were observed in rh2 and sws2. The rh2-a, one of the three subtypes of rh2, was absent in the genome of V. variegatus and pseudogenized in V. moseri. Moreover, rh2-a mRNA was not detected in M. achne and P. olivaceus, despite the presence of a functional reading frame. Analyses of the maximum absorption spectra (λmax) estimated by in vitro reconstitution indicated that SWS2A of M. achne (451.9 nm) and P. olivaceus (465.6 nm) were blue-sensitive, whereas in V. variegatus (485.4 nm), it was green-sensitive and comparable to V. moseri (482.3 nm). Our results indicate that although the four flounder species possess a similar opsin gene repertoire, the SWS2A opsin of the genus Verasper is functionally green-sensitive, while its overall structure remains conserved as a blue-sensitive opsin. Further, the rh2-a function seems to have been reduced during the evolution of flounders. λmax values of predicted ancestral SWS2A of Pleuronectiformes and Pleuronectidae was 465.4 and 462.4 nm, respectively, indicating that these were blue-sensitive. Thus, the green-sensitive SWS2A is estimated to be arisen in ancestral Verasper genus. It is suggested that the sensitivity shift of SWS2A from blue to green may have compensated functional reduction in RH2-A.

Effects of different green light intensities on the growth performance and endocrine properties of barfin flounder Verasper moseri.

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.

Expression of genes for melanotropic peptides and their receptors for morphological color change in goldfish Carassius auratus.

To evaluate the association of the melanotropic peptides and their receptors for morphological color change, we investigated the effects of changes in background color, between white and black, on xanthophore density in the scales and expression levels of genes for hormonal peptides and corresponding receptors (MCH-R2, MC1R, and MC5R) in goldfish (Carassius auratus). The xanthophore density in both dorsal and ventral scales increased after transfer from a white to black background. However, xanthophore density in dorsal scales increased after transfer from a black to white background, and that of ventral scales decreased after transfer from a black to black background, which served as the control. In the white-reared fish, melanin-concentrating hormone (mch) mRNA content in the brain was higher than that in black-reared fish, whereas proopiomelanocortin a (pomc-a) mRNA content in the pituitary was lower than that in the black-reared fish. Agouti-signaling protein (asp) mRNA was detected in the ventral skin but not in the dorsal skin. No difference was observed in the asp mRNA content between fish reared in white or black background, suggesting that ASP might not be associated with background color adaptation. In situ hybridization revealed that both mc1r and mc5r were expressed in the xanthophores in scales. The mRNA content of mc1r in scales did not always follow the background color change, whereas those of mc5r decreased in the white background and increased in the black background, suggesting that mc5r might be a major factor reinforcing the function of MSH in morphological color changes. White backgrounds increased mch mRNA content in the brain, but decreased mch-r2 mRNA content in the scales. These altered expression levels of melanotropin receptors might affect reactivity to melanotropins through long-term adaptation to background color.

Data on the density of xanthophores in a whole scale of goldfish acclimated to white or black background color.

The data presented in this article are related to the research article entitled "Expression of genes for melanotropic peptides and their receptors for morphological color change in goldfish Carassius auratus" (Mizusawa et al., In press) [1]. This article describes data on the density of xanthophores in the scales of goldfish acclimated to white or black background color. To determine the effects of acclimation history during long-term background color adaptation, fish were transferred from a white tank to a white or black tank and vice versa halfway through the acclimation process. To observe xanthophores, the iridophore layer was scraped from the scale and the pteridine/carotenoid pigments were aggregated. The number of xanthophores was calculated after image processing.

Data for amino acid alignment of Japanese stingray melanocortin receptors with other gnathostome melanocortin receptor sequences, and the ligand selectivity of Japanese stingray melanocortin receptors.

This article contains structure and pharmacological characteristics of melanocortin receptors (MCRs) related to research published in "Characterization of melanocortin receptors from stingray Dasyatis akajei, a cartilaginous fish" (Takahashi et al., 2016) [1]. The amino acid sequences of the stingray, D. akajei, MC1R, MC2R, MC3R, MC4R, and MC5R were aligned with the corresponding melanocortin receptor sequences from the elephant shark, Callorhinchus milii, the dogfish, Squalus acanthias, the goldfish, Carassius auratus, and the mouse, Mus musculus. These alignments provide the basis for phylogenetic analysis of these gnathostome melanocortin receptor sequences. In addition, the Japanese stingray melanocortin receptors were separately expressed in Chinese Hamster Ovary cells, and stimulated with stingray ACTH, α-MSH, ß-MSH, γ-MSH, δ-MSH, and ß-endorphin. The dose response curves reveal the order of ligand selectivity for each stingray MCR.

Characterization of melanocortin receptors from stingray Dasyatis akajei, a cartilaginous fish.

Melanocortin (MC) systems are composed of MC peptides such as adrenocorticotropic hormone (ACTH), several molecular forms of melanocyte-stimulating hormones (MSHs) and MC receptors (MCRs). Here we demonstrated that the cartilaginous fish, Dasyatis akajei (stingray) expresses five subtypes of MCR genes-mc1r to mc5r-as in the case of teleost and tetrapod species. This is the first evidence showing the presence of the full repertoire of melanocortin receptors in a single of cartilaginous fish. Expression of respective stingray mcr cDNAs in Chinese hamster ovary cells revealed that Des-acetyl-α-MSH exhibited cAMP-producing activity indistinguishable to ACTH(1-24) on MC1R and MC2R, while the activity of Des-acetyl-α-MSH on MC3R, MC4R, and MC5R were similar to or slightly greater than that of ACTH(1-24). Notably, in contrast to the other vertebrates, MC2R did not require coexpression with a melanocortin receptor-2 accessory protein 1 (mrap1) cDNA for functional expression. One of the roles of MC system resides in regulation of the pituitary-interrenal (PI) axis-a homologue of tetrapod pituitary-adrenal axis. In stingray, interrenal tissues were shown to express mc2r and mc5r as major MCR genes. These results established the presence of functional PI axis in stingray at the level of receptor molecule. While MC2R participates in adrenal functions together with MRAP1 in tetrapod species, the fact that sensitivity of MC5R to Des-acetyl-α-MSH and ACTH(1-24) were two order of magnitude higher than MC2R without coexpression with MRAP1 suggested that MC5R could play a more important role than MC2R to transmit signals conveyed by ACTH and MSHs if MRAP1 is really absent in the stingray.

Chronic effects of light irradiated from LED on the growth performance and endocrine properties of barfin flounder Verasper moseri.

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.

Involvement of melanin-concentrating hormone 2 in background color adaptation of barfin flounder Verasper moseri.

In teleosts, melanin-concentrating hormone (MCH) plays a key role in skin color changes. MCH is released into general circulation from the neurohypophysis, which causes pigment aggregation in the skin chromatophores. Recently, a novel MCH (MCH2) precursor gene, which is orthologous to the mammalian MCH precursor gene, has been identified in some teleosts using genomic data mining. The physiological function of MCH2 remains unclear. In the present study, we cloned the cDNA for MCH2 from barfin flounder, Verasper moseri. The putative prepro-MCH2 contains 25 amino acids of MCH2 peptide region. Liquid chromatography-electrospray ionization mass spectrometry with a high resolution mass analyzer were used for confirming the amino acid sequences of MCH1 and MCH2 peptides from the pituitary extract. In vitro synthesized MCH1 and MCH2 induced pigment aggregation in a dose-dependent manner. A mammalian cell-based assay indicated that both MCH1 and MCH2 functionally interacted with both the MCH receptor types 1 and 2. Mch1 and mch2 are exclusively expressed in the brain and pituitary. The levels of brain mch2 transcript were three times higher in the fish that were chronically acclimated to a white background than those acclimated to a black background. These results suggest that in V. moseri, MCH1 and MCH2 are involved in the response to changes in background colors, during the process of chromatophore control.

Molecular and functional characterization of opsins in barfin flounder (Verasper moseri).

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.