Sung Pyo Hur

Fish sleeping under sandy bottom: Interplay of melatonin and clock genes

Wrasse species exhibit a definite daily rhythm in locomotor activity and bury themselves in the sand at the bottom of the ocean at night. It remains unclear how their behavior in locomotor activity is endogenously regulated. The aim of the present study was to clarify the involvement of melatonin and clock genes (Per1, Per2, Bmal1, and Cry1) in daily and circadian rhythms of the threespot wrasse, Halichoeres trimaculatus, which is a common species in coral reefs. Daily and circadian rhythms in locomotor activity were monitored under conditions of light-dark cycle (LD=12:12), constant light (LL), and darkness (DD). Daily rhythms in locomotor activity were observed under LD and persisted under LL and DD. Melatonin from a cultured pineal gland showed daily variations with an increase during the nighttime and a decrease during daytime, which persisted under DD. Melatonin treatment induced decreases in locomotor activity and respiratory rate, suggesting that melatonin has a sleep-inducing effect. Per1 and Per2 mRNA abundance in the brain under LD showed daily rhythms with an increase around lights on. Robust oscillation of Per1 and Per2 mRNA expression persisted under DD and LL, respectively. Expression of Bmal1 and Cry1 mRNA also showed daily and circadian patterns. These results suggest that clock genes are related to circadian rhythms in locomotor activity and that melatonin plays a role in inducing a sleep-like state after fish bury themselves in the sand. We conclude that the sleep-wake rhythm of the wrasse is regulated by a coordination of melatonin and clock genes.

Lunar phase-dependent expression of cryptochrome and a photoperiodic mechanism for lunar phase-recognition in a reef fish, goldlined spinefoot.

Lunar cycle-associated physiology has been found in a wide variety of organisms. Recent study has revealed that mRNA levels of Cryptochrome (Cry), one of the circadian clock genes, were significantly higher on a full moon night than on a new moon night in coral, implying the involvement of a photoreception system in the lunar-synchronized spawning. To better establish the generalities surrounding such a mechanism and explore the underlying molecular mechanism, we focused on the relationship between lunar phase, Cry gene expression, and the spawning behavior in a lunar-synchronized spawner, the goldlined spinefoot (Siganus guttatus), and we identified two kinds of Cry genes in this animal. Their mRNA levels showed lunar cycle-dependent expression in the medial part of the brain (mesencephalon and diencephalon) peaking at the first quarter moon. Since this lunar phase coincided with the reproductive phase of the goldlined spinefoot, Cry gene expression was considered a state variable in the lunar phase recognition system. Based on the expression profiles of SgCrys together with the moonlights pattern of timing and duration during its nightly lunar cycle, we have further speculated on a model of lunar phase recognition for reproductive control in the goldlined spinefoot, which integrates both moonlight and circadian signals in a manner similar to photoperiodic response.

Impacts of moonlight on fish reproduction.

The waxing and waning cycle of the moon is repeated at approximately 1-month intervals, and concomitant changes occur in the levels of moonlight and cueing signals detected by organisms on the earth. In the goldlined spinefoot Siganus guttatus, a spawner lunar-synchronized around the first quarter moon, periodic changes in moonlight are used to cue gonadal development and gamete release. Rearing of mature fish under artificial constant full moon and new moon conditions during the spawning season leads to disruption or delay of synchronous spawning around the predicted moon phase. Melatonin, an endogenous transducer of the environmental light/dark cycle, increases in the blood and in the pineal gland around the new moon period and decreases around the full moon period. In synchrony with melatonin fluctuation, melatonin receptor(s) mRNA abundance is higher during the new moon period than during the full moon. The melatonin/melatonin receptor system is likely affected by moonlight. Measurements of the expression patterns of clock genes in neural tissues demonstrate that Cryptochrome (Cry1 and Cry3) and Period (Per2) fluctuate with lunar periodicity, the former peaking in the medial part of the brain around the first quarter moon period, and the latter peaking in the pineal gland around the full moon. Some clock genes may respond to periodic changes in moon phase and appear to be involved in the generation of lunar-related rhythmicity in lunar spawners. Thus, some fish use moonlight-related periodicities as reliable information for synchronizing the timing of reproductive events.

Effects of different colors of light on melatonin suppression and expression analysis of Aanat1 and melanopsin in the eye of a tropical damselfish

Ocular melatonin production exhibits a daily rhythm with a decrease during photophase and an increase during scotophase (nocturnal pattern) in teleost fish due to day-night changes in the activity of the rate-limiting melatonin synthesizing enzyme arylalkylamine N-acetyltransferase (AANAT). Acute light exposure during scotophase suppresses AANAT activity and melatonin production in the eyes, suggesting that external light signals are a principal regulator of ocular melatonin synthesis. To better understand the photic regulation of ocular melatonin synthesis in teleost fish, this study sought to characterize the effect of light on ocular melatonin synthesis in the sapphire devil Chrysiptera cyanea, which shows a nocturnal pattern and light-induced inhibition of ocular melatonin production during scotophase. Exposure to three different wavelengths of light (half-peak bandwidth=435-475 nm with a peak of 455 nm, 495-565 nm with a peak of 530 nm, and 607-647 nm with a peak of 627 nm for the blue, green, and red LEDs) for 2h during scotophase resulted in the blue wavelength significantly decreasing ocular melatonin content within 30 min after light exposure. This result clearly indicates that the effective range of visible light on ocular melatonin suppression is distributed within the wavelengths of blue light and that a blue light-sensitive opsin is involved in ocular melatonin suppression in the fish. A PCR-based cloning method revealed the expression of melanopsin, a putative blue light-sensitive nonvisual opsin, in the eyes. Furthermore, in situ hybridization using the sapphire devil Aanat1 and melanopsin RNA probes showed mRNA expressions of both genes in the inner nuclear and ganglion cell layer of the fish retina. These results suggest that melanopsin is a possible candidate photoreceptor involved in ocular melatonin suppression by an external light signal in the sapphire devil.

Molecular cloning and expression of long-wavelength-sensitive cone opsin in the brain of a tropical damselfish

Ovarian development of the sapphire devil, Chrysiptera cyanea, exhibits photoperiodism and is stimulated under long-day conditions. Previous studies suggest that red light is more effective than green and blue lights for inducing ovarian development. In addition, the extra-retinal photoreception involved in the ovarian development is suggested in this species. The present study aimed to clone the red-light-sensitive cone opsin (sdLWS) of this species, to demonstrate its expression in the brain, and to confirm the effectiveness of red light on the initiation of ovarian development. A phylogenetic analysis revealed that sdLWS belongs to the long-wavelength-sensitive opsin (LWS opsin) group, showing high identity (77-92%) with the LWS opsin of other fishes. RT-PCR showed that sdLWS is expressed in the eye, brain, and ovary. In situ hybridization indicated that sdLWS is expressed in the third ventricle periventricular area in the anterior hypothalamus. Exposing fish to long-day conditions of red light resulted in the appearance of vitellogenic oocytes in the ovary and an increase in the gonadosomatic index within 2weeks, suggesting that fish immediately undergo oocyte development under red light conditions. These results indicate that sdLWS is a possible candidate of deep brain photoreceptor molecule involved in photoperiodic ovarian development in the sapphire devil.

Influence of moonlight on mRNA expression patterns of melatonin receptor subtypes in the pineal organ of a tropical fish.

The goldlined spinefoot, Siganus guttatus, is a lunar-synchronized spawner, which repeatedly releases gametes around the first quarter moon during the reproductive season. A previous study reported that manipulating moonlight brightness at night disrupted synchronized spawning, suggesting involvement of this natural light source in lunar synchronization. The present study examined whether the mRNA expression pattern of melatonin receptor subtypes MT1 and Mel1c in the pineal organ of the goldlined spinefoot is related to moonlight. Real-time quantitative polymerase chain reaction analysis revealed that the abundance of MT1 and Mel1c mRNA at midnight increased during the new moon phase and decreased during the full moon phase. Exposing fish to moonlight intensity during the full moon period resulted in a decrease in Mel1c mRNA abundance within 1h. Fluctuations in the melatonin receptor genes according to changes in the moon phase agreed with those of melatonin levels in the blood. These results indicate that periodic changes in cues from the moon influence melatonin receptor mRNA expression levels. The melatonin-melatonin receptor system may play a role in predicting the moon phase through changes in night brightness.

Moonlight affects mRNA abundance of arylalkylamine N‐acetyltransferase in the retina of a lunar‐synchronized spawner, the goldlined spinefoot

Melatonin synthesis in the pineal gland and retina shows a rhythmic fashion with high levels at night and is controlled by a rate-limiting enzyme, arylalkylamine N-acetyltransferase (AANAT). A previous study revealed that moonlight suppresses the plasma melatonin levels of the goldlined spinefoot (Siganus guttatus), which exhibits a lunar cycle in its reproductive activity and repeats gonadal development toward and spawning around the first quarter moon. Whether the retina of this species responds to moonlight is unknown. To clarify the photoperceptive ability of this species, we aimed to clone the full-length cDNA of Aanat1 (sgAanat1) from the retina and examine its transcriptional pattern under several daylight and moonlight regimes. The full-length sgAanat1 cDNA (1,038 bp) contained a reading frame encoding a protein of 225 amino acids, which was highly homologous to AANAT1 of other teleosts. Reverse transcription-polymerase chain reaction (PCR) analysis revealed that among the tissues tested, sgAanat1 fragments were expressed exclusively in the retina. Real-time quantitative PCR analysis revealed that sgAanat1 fluctuated with high abundance at night under light-dark cycle and at subjective night under constant darkness, but not under constant light. These results suggest that sgAanat1 is regulated by both the external light signal and internal clock system. The abundance of sgAanat1 in the retina was higher at the culmination time around new moon than full moon phase. Additionally, exposing fish to brightness around the full moon period suppressed sgAanat1 mRNA abundance. Thus, moonlight is perceived by fish and has an impact on melatonin fluctuation in the retina.

Molecular cloning of kisspeptin receptor genes (gpr54-1 and gpr54-2) and their expression profiles in the brain of a tropical damselfish during different gonadal stages

The kisspeptin receptor (GPR54) mediates neuroendocrine control of kisspeptin in the brain and acts as a gateway for a pulsatile release of hypothalamic gonadotropin-releasing hormone. This study aimed to clone two GPR54 genes (gpr54-1 and gpr54-2) from the brain of the sapphire devil Chrysiptera cyanea, a tropical damselfish, and to study their involvement in reproduction. The partial sequences of the sapphire devil gpr54-1 cDNA (1059bp) and gpr54-2 cDNA (1098bp) each had an open reading frame encoding a protein of 353 and 366 amino acids, respectively, both of which had structural features of a G-protein-coupled receptor. The expression of gpr54-1 mRNA was observed in the diencephalon and telencephalon, and gpr54-2 mRNA was found in the optic tectum of sapphire devil. When gpr54-1 and gpr54-2 mRNA levels were examined in the brain of sapphire devil by real-time quantitative polymerase chain reaction (qPCR), they were found to increase during late vitellogenesis and post-spawning. Treatment of fish with estradiol-17β (Ε2) resulted in an increase in gpr54-1 and gpr54-2 expression in the brain of sapphire devil. Thus, kisspeptin receptors likely mediate the activity of kisspeptin in the brain and are involved in controlling reproductive events in a tropical damselfish.

Effect of Light Intensity and Wavelength on Diurnal Activity of the Banded Coral Shrimp Stenopus hispidus (Decapoda, Stenopodidae): A Possible Adaptation for a Cleaner Shrimp in Reef Environments

Abstract: Most decapod crustaceans are nocturnal. However, because cleaner shrimp at cleaning stations act in concert with their hosts, they clean debris and parasites off the body surfaces of diurnal hosts during daytime. It is not known how cleaner shrimp physiologically accommodate diurnal environments and perform cleaning behaviors. We examined the effect of light cues on the daily rhythmicity of the banded coral shrimp Stenopus hispidus (Decapoda, Stenopodidae), a cleaner shrimp in coral reefs. Shrimp were individually kept in aquaria under conditions of 12-hr light and 12-hr darkness (LD) and constant darkness (DD). Double-plotted actogram analyses revealed that shrimp under LD were active during scotophase and inactive during photophase. Locomotor rhythms were observed in most shrimp under DD. Periodogram analyses showed a weak circadian rhythm in shrimp under DD. Our results show that this species is nocturnal and that its locomotor activity is controlled primarily by the LD cycle. Day-night differences in locomotor activity were reduced in shrimp under LD with weak irradiance, and they remained active during photophase. Shrimp under LD using blue or green light-emitting diode light, but not red light-emitting diode light, showed a day-inactive and night-active rhythmicity. These results indicate that this species can be active under environments with low green and blue spectra—for example, during twilight hours, at depth, or on cloudy days—even during daytime, and this weakness of the circadian clock may be advantageous in their role as cleaner shrimp. It is concluded that in addition to the presence of visiting hosts, light conditions at cleaning stations are likely to influence cleaning activity.

Existence of a photoinducible phase for ovarian development and photoperiod-related alteration of clock gene expression in a damselfish

The sapphire devil, Chrysiptera cyanea, is a reef-associated damselfish and their ovarian development can be induced by a long photoperiod. In this study, we demonstrated the existence of a photoinducible phase for the photoperiodic ovarian development in the sapphire devil. Induction of ovarian development under night-interruption light schedules and Nanda-Hamner cycles revealed that the photoinducible phase appeared in a circadian manner between ZT12 and ZT13. To characterize the effect of photoperiod on clock gene expression in the brain of this species, we determined the expression levels of the sdPer1, sdPer2, sdCry1, and sdCry2 clock genes under constant light and dark conditions (LL and DD) and photoperiodic (short and long photoperiods). The expression of sdPer1 exhibited clear circadian oscillation under both LL and DD conditions, while sdPer2 and sdCry1 expression levels were lower under DD than under LL conditions and sdCry2 expression was lower under LL than under DD conditions. These results suggest a key role for sdPer1 in circadian clock cycling and that sdPer2, sdCry1, and sdCry2 are light-responsive clock genes in the sapphire devil. After 1 week under a long photoperiod, we observed photoperiod-related changes in sdPer1, sdPer2, and sdCry2 expression, but not in sdCry1 expression. These results suggest that the expression patterns of some clock genes exhibit seasonal variation according to seasonal changes in day length and that such seasonal alteration of clock gene expression may contribute to seasonal recognition by the sapphire devil.