The Correlation between the Severity of Obstructive Sleep Apnea and Insulin Resistance in a Japanese Population.

Background: Repetitive episodes of apnea and hypopnea during sleep in patients with obstructive sleep apnea (OSA) are known to increase the risk of atherosclerosis. Underlying obesity and related disorders, such as insulin resistance, are indirectly related to the development of atherosclerosis. In addition, OSA is independently associated with insulin resistance; however, data regarding this relationship are scarce in Japanese populations. Methods: This study aimed to examine the relationship between the severity of OSA and insulin resistance in a Japanese population. We analyzed the data of consecutive patients who were referred for polysomnography under clinical suspicion of developing OSA and who did not have diabetes mellitus or any cardiovascular disease. Multiple regression analyses were performed to determine the relationship between the severity of OSA and insulin resistance. Results: The data from a total of 483 consecutive patients were analyzed. The median apnea-hypopnea index (AHI) was 40.9/h (interquartile range: 26.5, 59.1) and the median homeostasis model assessment for insulin resistance (HOMA-IR) was 2.00 (interquartile range: 1.25, 3.50). Multiple regression analyses revealed that the AHI, the lowest oxyhemoglobin saturation (SO2), and the percentage of time spent on SO2 < 90% were independently correlated with HOMA-IR (an adjusted R-squared value of 0.01278821, p = 0.014; an adjusted R-squared value of -0.01481952, p = 0.009; and an adjusted R-squared value of 0.018456581, p = 0.003, respectively). Conclusions: The severity of OSA is associated with insulin resistance assessed by HOMA-IR in a Japanese population.

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.

Cross-Sectional Relationship Between Atrial Conduction Delay and Arterial Stiffness in Patients with Obstructive Sleep Apnea.

Aim: Prolonged P-wave duration (PWD), which indicates atrial conduction delay, is a potent precursor of atrial fibrillation (AF) that may be induced by obstructive sleep apnea (OSA). The cardio-ankle vascular index (CAVI), which is an arterial stiffness parameter, is elevated in patients with OSA; moreover, an increased CAVI is associated with atrial conduction delay through left atrium enlargement in association with left ventricular diastolic dysfunction. We aimed to examine the relationship between the CAVI and PWD in patients with OSA. Methods: We included patients with a sinus rhythm who underwent overnight polysomnography. We measured the PWD and CAVI on standard 12-lead electrocardiograms; further, we analyzed the relationship between PWD and CAVI. Results: We analyzed data from 300 participants (men, 89.0%; mean age, 52.3 ± 13.1 years; and body mass index, 26.2 ± 3.9 kg/m2). The mean PWD was 104.4 ± 10.4 ms while the mean CAVI was 7.5 ± 1.5. PWD was significantly correlated with CAVI (r = 0.478, p < 0.001); additionally, PWD and CAVI were directly associated with OSA severity (p = 0.002 and p = 0.002, respectively). Multivariate regression analysis revealed an independent significant correlation of PWD and CAVI with OSA severity. Conclusion: In patients with OSA, an increase in arterial stiffness is associated with atrial conduction delay.

Clinical outcomes of chronic heart failure patients with unsuppressed sleep apnea by positive airway pressure therapy.

Introduction: Heart failure (HF) is an advanced stage of cardiac disease and is associated with a high rate of mortality. Previous studies have shown that sleep apnea (SA) is associated with a poor prognosis in HF patients. Beneficial effects of PAP therapy that is effective on reducing SA on cardiovascular events, were not yet established. However, a large-scale clinical trial reported that patients with central SA (CSA) which was not effectively suppressed by continuous positive airway pressure (CPAP) revealed poor prognosis. We hypothesize that unsuppressed SA by CPAP is associated with negative consequences in patients with HF and SA, including either obstructive SA (OSA) or CSA. Methods: This was a retrospective observational study. Patients with stable HF, defined as left ventricular ejection fraction of ≤50%; New York Heart Association class ≥ II; and SA [apnea-hypopnea index (AHI) of ≥15/h on overnight polysomnography], treated with CPAP therapy for 1 month and performed sleep study with CPAP were enrolled. The patients were classified into two groups according to AHI on CPAP (suppressed group: residual AHI ≥ 15/h; and unsuppressed group: residual AHI < 15/h). The primary endpoint was a composite of all-cause death and hospitalization for HF. Results: Overall, data of 111 patients including 27 patients with unsuppressed SA, were analyzed. The cumulative event-free survival rates were lower in the unsuppressed group during a period of 36.6 months. A multivariate Cox proportional hazard model showed that the unsuppressed group was associated with an increased risk for clinical outcomes (hazard ratio 2.30, 95% confidence interval 1.21-4.38, p = 0.011). Conclusion: Our study suggested that in patients with HF and SA including either OSA or CSA, presence of unsuppressed SA even on CPAP was associated with worse prognosis as compared to those with suppressed SA by CPAP.

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.

Sleep Apnea and Abnormal Respiratory Patterns with Deep Sedation during Radiofrequency Catheter Ablation in Patients with Atrial Fibrillation.

Background: Abnormal respiration during radiofrequency catheter ablation (RFCA) with deep sedation in patients with atrial fibrillation (AF) can affect the procedure's success. However, the respiratory pattern during RFCA with deep sedation remains unclear. This study aimed to investigate abnormal respiration during RFCA and its relationship with sleep apnea in patients with AF. Methods: We included patients with AF who underwent RFCA with cardiorespiratory monitoring using a portable polygraph both at night and during RFCA with deep sedation. The patients were divided based on the administered sedative medicines. Results: We included 40 patients with AF. An overnight sleep study revealed that 27 patients had sleep apnea; among them, 9 showed central predominance. During RFCA with deep sedation, 15 patients showed an abnormal respiratory pattern, with 14 patients showing obstructive predominance. Further, 17 and 23 patients were administered with propofol alone and dexmedetomidine plus propofol, respectively. There was no significant between-group difference in the respiratory event index (REI) at night (7.9 vs. 9.3, p = 0.744). However, compared with the group that received dexmedetomidine plus propofol, the propofol-alone group showed a higher REI during RFCA (5.4 vs. 2.6, p = 0.048), more frequent use of the airway (47% vs. 13%, p = 0.030), and a higher dose of administered propofol (3.9 mg/h/kg vs. 1.2 mg/h/kg, p < 0.001). Multivariable analysis revealed that only the propofol amount was associated with REI during RFCA (p = 0.007). Conclusions: Our findings demonstrated that respiratory events during RFCA with deep sedation were mainly obstructive. Propofol should be administered with dexmedetomidine rather than alone to reduce the propofol amount and avoid respiratory instability.

Evaluation of the Apnea-Hypopnea Index Determined by Adaptive Servo-Ventilation Devices in Patients With Heart Failure and Sleep-Disordered Breathing.

Introduction: Adaptive servo-ventilation (ASV) devices are designed to suppress central respiratory events, and therefore effective for sleep-disordered breathing (SDB) in patients with heart failure (HF) and provide information about their residual respiratory events. However, whether the apnea-hypopnea index (AHI), determined by the ASV device AutoSet CS (ASC), correlates with the AHI calculated by polysomnography (PSG) in patients with HF and SDB remains to be evaluated. Methods: Consecutive patients with SDB titrated on ASC were included in the study. We assessed the correlation between AHI determined by manual scoring during PSG (AHI-PSG) and that determined by the ASC device (AHI-ASC) during an overnight session. Results: Thirty patients with HF and SDB (age, 68.8 ± 15.4 years; two women; left ventricular ejection fraction, 53.8 ± 17.9%) were included. The median AHI in the diagnostic study was 28.4 events/h, including both obstructive and central respiratory events. During the titration, ASC markedly suppressed the respiratory events (AHI-PSG, 3.3 events/h), while the median AHI-ASC was 12.8 events/h. We identified a modest correlation between AHI-PSG and AHI-ASC (r = 0.36, p = 0.048). The Brand-Altman plot indicated that the ASC device overestimated the AHI, and a moderate agreement was observed with PSG. Conclusions: There was only a modest correlation between AHI-PSG and AHI-ASC. The discrepancy may be explained by either the central respiratory events that occur during wakefulness or the other differences between PSG and ASC in the detected respiratory events. Therefore, clinicians should consider this divergence when assessing residual respiratory events using ASC.

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.

Serum Neutrophil Gelatinase-associated Lipocalin (NGAL) Is Elevated in Patients with Asthma and Airway Obstruction.

Neutrophilic airway inflammation is one of the features of severe asthma. Neutrophil gelatinase-associated lipocalin (NGAL), or lipocalin-2, is a glycoprotein associated with neutrophilic inflammation and can be detected in blood. Recently, blood NGAL levels have been reported to be elevated in chronic obstructive pulmonary disease. However, the clinical significance of serum NGAL levels in patients with asthma has not been elucidated. The aim of this study was to explore the association between serum NGAL level and clinical parameters in patients with asthma. Sixty-one non-smoking people with stable asthma were enrolled in this study. All patients underwent blood collection and pulmonary function tests. The associations between serum NGAL levels and clinical parameters were analyzed retrospectively. Serum NGAL levels in patients with asthma and obstructive ventilatory defect were higher than those in patients with asthma without obstructive ventilatory defect (76.4±51.4 ng/mL vs. 39.3±27.4 ng/mL, P=0.0019). Serum NGAL levels were correlated with forced expired flow at 50% of vital capacity %predicted and forced expired flow at 75% of vital capacity %predicted (r=-0.3373, P=0.0078 and r=-0.2900, P=0.0234, respectively). Results of a multiple regression analysis demonstrated that serum NGAL level was independently associated with obstructive ventilatory defect. Serum NGAL levels were elevated in patients with asthma and obstructive ventilatory defect. NGAL may be involved in airway remodeling possibly mediated by neutrophilic inflammation in asthma.

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.

Clinical utility of a type 4 portable device for in-home screening of sleep disordered breathing.

BACKGROUND: Portable monitoring devices have been developed for in-home screening and to aid in the diagnosis of sleep disordered breathing (SDB) while increasing accessibility and reducing costs. Although there are many different devices available in the market, most have not undergone rigorous validation. Therefore, although such devices are promising, more research on their clinical utility is necessary. The purpose of this study was to assess the clinical utility of a type 4 home sleep apnea test (HSAT) as an in-home screening for SDB. METHODS: We investigated consecutive subjects who underwent in-laboratory overnight polysomnography following in-home screening using HSAT. We evaluated the correlation between apnea-hypopnea index (AHI) by in-laboratory overnight polysomnography and by HSAT and evaluated the sensitivity and specificity for AHI ≥5 and AHI ≥30 by the receiver operating characteristic (ROC) analysis. RESULTS: Finally, data of 387 participants (86.8% men, mean age 55.3±13.3 years and body mass index 25.1±4.1 kg/m2) were assessed. In all patients, AHI by HSAT correlated significantly with AHI by polysomnography (r=0.670, P<0.001). The area under curves of ROC for AHI ≥5 and AHI ≥30 were 0.854±0.029 and 0.841±0.022, respectively. The best cut-off of AHI by HSAT for detecting AHI by polysomnography ≥5 was 10.3 events/h (sensitivity, 82.8%; and specificity, 76.0%), and AHI by HSAT for detecting AHI by polysomnography ≥30 was 24.5 events/h (sensitivity, 75.8%; and specificity, 80.4%). CONCLUSIONS: This type 4 HSAT may have potential as a screening tool for SDB and thus have sufficient clinical utility.

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.

A new targeted capture method using bacterial artificial chromosome (BAC) libraries as baits for sequencing relatively large genes.

To analyze a specific genome region using next-generation sequencing technologies, the enrichment of DNA libraries with targeted capture methods has been standardized. For enrichment of mitochondrial genome, a previous study developed an original targeted capture method that use baits constructed from long-range polymerase chain reaction (PCR) amplicons, common laboratory reagents, and equipment. In this study, a new targeted capture method is presented, that of bacterial artificial chromosome (BAC) double capture (BDC), modifying the previous method, but using BAC libraries as baits for sequencing a relatively large gene. We applied the BDC approach for the 214 kb autosomal region, ring finger protein 213, which is the susceptibility gene of moyamoya disease (MMD). To evaluate the reliability of BDC, cost and data quality were compared with those of a commercial kit. While the ratio of duplicate reads was higher, the cost was less than that of the commercial kit. The data quality was sufficiently the same as that of the kit. Thus, BDC can be an easy, low-cost, and useful method for analyzing individual genome regions with substantial length.

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.

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.