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.

A surge in respiratory syncytial virus infection-related hospitalizations associated with the COVID-19 pandemic: An observational study at pediatric emergency referral hospitals in Tokushima Prefecture.

The outbreak of coronavirus disease (COVID-19) resulted in implementation of social distancing and other public health measures to control the spread of infection and improve prevention, resulting in a decrease in respiratory syncytial virus (RSV) and pediatric respiratory tract infection rates. However, there was a rapid and large re-emergence of RSV infection in Japan. Notably, we were faced with a difficult situation wherein there was a shortage of hospital beds. This study aimed to examine the epidemiological patterns of RSV-related hospitalizations among children before and after the COVID-19 pandemic onset at two pediatric emergency referral hospitals covering the entire Tokushima Prefecture. Data were extracted from electronic medical records of children hospitalized with RSV infection between January 1, 2018, and December 31, 2021. All patients meeting the eligibility criteria were included in this study. The rates of study outcomes were documented annually during 2018-2021 and compared between the 2018-2020 and 2021 periods. In 2020, there was no RSV infection outbreak. Hospitalizations at the peak week in 2021 were 2.2- and 2.8-fold higher than those in 2018 and 2019, respectively. Hospitalizations in 2021 were concentrated within a short period. In addition, there was a significant increase in hospitalizations among children aged 3-5 months and those older than 24 months. The high-flow nasal cannula (HFNC) use rate nearly doubled in 2021. A new pandemic in the future may cause an outbreak of RSV infection that can result in an intensive increase in the number of hospitalizations of pediatric patients requiring respiratory support, especially in infants aged <6 months. There is an urgent need to improve the preparedness of medical systems, particularly in terms of the number of inpatient beds and the immediate availability of HFNC.

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.

Left-right pigmentation pattern of Japanese flounder corresponds to expression levels of melanocortin receptors (MC1R and MC5R), but not to agouti signaling protein 1 (ASIP1) expression.

Body coloration in flatfish is one of the most distinctive asymmetries in the animal kingdom, although the fundamental molecular mechanism of the pigmentation is unclear. In the dorso-ventral coloration (countershading) of other teleost fishes, ventral-specific expression of agouti signaling protein 1 (ASIP1), an endogenous antagonist of melanocortin 1 receptor (MC1R), has been reported to play a pivotal role. Contribution of ASIP1 is also suggested in the asymmetrical pigmentation of flatfish. In order to confirm the contribution of ASIP1 and further examine receptor function in the body coloration of Japanese flounder, expression levels of asip1, mc1r, melanocortin 5 receptor (mc5r), and melanin-concentrating hormone receptor 2 (mchr2) were measured in the normally pigmented area of the left side, the normally non-pigmented area of the right side, and the abnormally pigmented (exhibiting hypermelanosis) area of the right side. Measurement was also carried out under conditions of hypermelanosis stimulated by cortisol and during the transition from non-pigmentation to pigmentation in areas of hypermelanosis. Contrary to our expectations, no difference was detected in asip1 expression between pigmented and non-pigmented areas. There was also no difference between normal and hormonally stimulated pigmented conditions in areas of hypermelanosis or during the transition process. Instead, the expression levels of mc1r, mc5r, and mchr2 were consistently higher in pigmented areas, and were especially increased under hormonally stimulated conditions. In addition, expressions of these receptor genes increased prior to pigmentation in areas of future hypermelanosis. Our results suggest that MC1Rand MC5R, but not necessarily ASIP1, contribute to pigmentation and hypermelanosis in Japanese flounder. We propose a yet unknown molecular mechanism for asymmetrical pigmentation in flatfish that is distinct from that of countershading in other vertebrates.

Evaluating the interactions between red stingray (Dasyatis akajei) melanocortin receptors and elephant shark (Callorhinchus milii) MRAP1 and MRAP2 following stimulation with either stingray ACTH(1-24) or stingray Des-Acetyl-αMSH: A pharmacological study in Chinese Hamster Ovary cells.

Previous studies on bony vertebrate MC2R orthologs (i.e., ray finned fishes, amphibians, reptiles, birds, and mammals) have shown that these MC2R orthologs have an obligatory requirement for interaction with bony vertebrate MRAP1 orthologs to a) allow for the trafficking of the MC2R ortholog to the plasma membrane; and b) to allow activation by ACTH, but not by any MSH-sized ligand. In addition, previous studies have found that co-expression of teleost and mammalian MC4R orthologs with corresponding MRAP2 has positive effects on sensitivity to stimulation by αMSH or ACTH. MRAP1 and MRAP2 paralogs have been detected in the genome of a cartilaginous fish (elephant shark), yet two cartilaginous fish MC2R orthologs (elephant shark and red stingray) do not apparently require MRAP1 for trafficking to the plasma membrane when expressed in Chinese Hamster Ovary (CHO) cells, and both orthologs can be activated by either ACTH or MSH-sized ligands. This study was done to determine whether sensitivity to stimulation by ACTH(1-24) or Des-Acetyl-αMSH is affected when stingray (sr) MC1R, MC2R, MC3R, MC4R or MC5R were co-expressed in CHO cells with either elephant shark (es) MRAP1 or esMRAP2. The results indicated that co-expression with heterologous MRAP1 increased the sensitivity of all five stingray melanocortin receptors for srACTH(1-24), but had not statistically significant effect on stimulation by srDes-Acetyl-αMSH for any of the stingray melanocortin receptors. Conversely, co-expression with esMRAP2 only enhanced sensitivity for srDes-Acetyl-αMSH for srMC4R, but had no effect on the other stingray orthologs, and there was no increase in sensitivity for srACTH(1-24) for any of the stingray melanocortin receptors. It appears then that some stingray melanocortin receptors have retained the ability to interact with a cartilaginous MRAP1 paralog. These results are discussed with reference to radiation of MRAP-related accessory proteins in cartilaginous fishes.

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.

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.

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.

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.

Spectral sensitivity of guppy visual pigments reconstituted in vitro to resolve association of opsins with cone cell types.

The guppy (Poecilia reticulata) shows remarkable variation of photoreceptor cells in the retina, especially those sensitive to middle-to-long wavelengths of light. Microspectrophotometry (MSP) has revealed varying "green", "green-yellow" and "yellow" cone cells among guppies in Trinidad and Venezuela (Cumana). In the guppy genome, there are four "long-wave" opsin loci (LWS-1, -2, -3 and -4). Two LWS-1 alleles have potentially differing spectral sensitivity (LWS-1/180Ser and LWS-1/180Ala). In addition, two "middle-wave" loci (RH2-1 and -2), two "short-wave" loci (SWS2-A and -B), and a single "ultraviolet" locus (SWS1) as well as a single "rhodopsin" locus (RH1) are present. However, the absorption spectra of these photopigments have not been measured directly and the association of cell types with these opsins remains speculative. In the present study, we reconstituted these opsin photopigments in vitro. The wavelengths of maximal absorbance (λmax) were 571nm (LWS-1/180Ser), 562nm (LWS-1/180Ala), 519nm (LWS-3), 516nm (LWS-2), 516nm (RH2-1), 476nm (RH2-2), 438nm (SWS2-A), 408nm (SWS2-B), 353nm (SWS1) and 503nm (RH1). The λmax of LWS-3 is much shorter than the value expected (560nm) from the "five-sites" rule. The two LWS-1 alleles could explain difference of the reported MSP λmax values for the yellow cone class between Trinidad and Cumana guppies. Absence of the short-wave-shifted LWS-3 and the green-yellow cone in the green swordtail supports the hypothesis that this cell class of the guppy co-expresses the LWS-1 and LWS-3. These results reveal the basis of variability in the guppy visual system and provide insight into the behavior and ecology of these tropical fishes.

Immunohistochemical detection of corticotropin-releasing hormone (CRH) in the brain and pituitary of the hagfish, Eptatretus burgeri.

The distribution of corticotropin-releasing hormone (CRH) in the brain and pituitary of the hagfish Eptatretus burgeri, representing the earliest branch of vertebrates, was examined by immunohistochemistry to better understand the neuroendocrine system of hagfish. CRH-immunoreactive (ir) cell bodies were detected in the preoptic nucleus, periventricular preoptic nucleus, infundibular nucleus of the hypothalamus, and in the nucleus "A" of Kusunoki et al. (1982) in the medulla oblongata. In the brain, CRH-ir fibers were detected in almost all areas except for the olfactory bulb and telencephalon. Bundles of CRH-ir fibers were detected in the dorsal wall of the neurohypophysis. However, CRH-ir fibers were distant from adrenocorticotropic hormone (ACTH) cells in the adenohypophysis, as studied by dual-label immunohistochemistry. Cortisol and corticosterone were detected in the plasma by a combination of reverse-phase high performance liquid chromatography and a time-resolved fluoroimmunoassay. These results suggest that in the hagfish, CRH, ACTH, and corticosteroids exist and that CRH released in the neurohypophysis likely reaches the adenohypophysis via diffusion.