Akihiro Takemura

Moonlight affects nocturnal Period2 transcript levels in the pineal gland of the reef fish Siganus guttatus

Abstract:u2002 The golden rabbitfish Siganus guttatus is a reef fish with a restricted lunar‐synchronized spawning cycle. It is not known how the fish recognizes cues from the moon and exerts moon‐related activities. In order to evaluate the perception and utilization of moonlight by the fish, the present study aimed to clone and characterize Period2 (Per2), a light‐inducible clock gene in lower vertebrates, and to examine daily variations in rabbitfish Per2 (rfPer2) expression as well as the effect of light and moonlight on its expression in the pineal gland. The partially‐cloned rfPer2 cDNA (2933 bp) was highly homologous (72%) to zebrafish Per2. The rfPer2 levels increased at ZT6 and decreased at ZT18 in the whole brain and several peripheral organs. The rfPer2 expression in the pineal gland exhibited a daily variation with an increase during daytime. Exposing the fish to light during nighttime resulted in a rapid increase of its expression in the pineal gland, while the level was decreased by intercepting light during daytime. Two hours after exposing the fish to moonlight at the full moon period, the rfPer2 expression was upregulated. These results suggest that rfPer2 is a light‐inducible clock gene and that its expression is affected not only by daylight but also by moonlight. Since the rfPer2 expression level during the full moon period was higher than that during the new moon period, the monthly variation in the rfPer2 expression is likely to occur with the change in amplitude between the full and new moon periods.

Molecular cloning and daily variations of the Period gene in a reef fish Siganus guttatus

As the first step in understanding the molecular oscillation of the circa rhythms in the golden rabbitfish Siganus guttatus—a reef fish with a definite lunar-related rhythmicity—we cloned and sequenced a Period gene (rfPer). The rfPer gene contained an open reading frame that encodes a protein consisting of 1,452 amino acids; this protein is highly homologous to PER proteins of vertebrates including zebrafish. Phylogenetic analyses indicated that the rfPER protein is related to the zebrafish PER1 and PER4. The expression of rfPer mRNA in the whole brain, retina, and liver under light/dark (LD) conditions increased at 06:00xa0h and decreased at 18:00xa0h, suggesting that its robust circadian rhythm occurs in neural and peripheral tissues. When daily variation in the expression in rfPer mRNA in the whole brain and cultured pineal gland were examined under LD conditions, similar expression patterns of the gene were observed with an increase around dawn. Under constant light condition, the increased expression of rfPer mRNA in the whole brain disappeared around dawn. The present results demonstrate that rfPer is related to zPer4 and possibly zPer1. The present study is the first report on the Period gene from a marine fish.

Effect of cortisol on melatonin production by the pineal organ of tilapia, Oreochromis mossambicus.

The aim of the present study was to examine the involvement of cortisol on melatonin synthesis in the pineal organ of the Mozambique tilapia, Oreochromis mossambicus. The circulating levels of melatonin in this species exhibited daily variations with a decrease during the photophase (0600, 1200, and 1800 h) and an increase during the scotophase (0000 h), while cortisol levels peaked during the early photophase (0600 h). The pineal organ was cultured in vitro in the dark in the presence of cortisol mimicking either stressed (100 ng/mL) or resting (10 ng/mL) concentration in tilapia. High cortisol concentration significantly reduced the levels of melatonin secreted into the medium. In the fish reared under stressful conditions, the nocturnal circulating levels of cortisol increased significantly, while melatonin did not change significantly. We detected glucocorticoid receptor (GR) transcripts in the pineal organ and a quantitative real-time PCR revealed that this receptor mRNA abundance fluctuated diurnally, increasing at 0600, 1800, and 0000 h and decreasing at 1200 h. The GR mRNA abundance in the pineal organ was not altered either in vitro when the organ was cultured in the presence of 100 ng/mL cortisol or in vivo when the fish were reared under stressful conditions. On the basis of these findings, it is proposed that cortisol lowers melatonin synthesis in the pineal organ, while the role of GR signaling in this process remains to be established.

Environmental control of gonadal development in the tropical damselfish Chrysiptera cyanea

Abstract Photoperiod and temperature are potent environmental factors regulating the reproductive cycle of temperate fish. It is unclear whether fish in the tropics utilize these environmental factors for initiation and termination of their reproductive activity. The aim of this study was to examine the involvement of photoperiod and temperature in ovarian development of sapphire devil, Chrysiptera cyanea, which is a reef-associated tropical damselfish. Monthly collections of individuals in the experimental region (26°42′N, 127°52′E) revealed an increase in the gonadosomatic index (GSI) and appearance of vitellogenic oocytes in ovaries from March to August. Post-ovulated follicles and yolk-laden oocytes coexisted in some ovaries from May to August. These results suggest that the sapphire devil in this region has a restricted reproductive season with repeated spawnings. Rearing the fish during the resting phase (December) under experimental conditions of a long photoperiod (LD 14:10) at 31°C for 15 days and at 25 or 31°C for 60 days resulted in a significant increase in GSI and induction of vitellogenic oocytes, whereas a short photoperiod (LD 10:14) at the same temperatures failed to induce oocytes at the yolk stages. Long-term rearing of the fish at 31°C caused regression of vitellogenic oocytes. During the reproductive season, the fish reared at 25°C had higher spawning frequency than those at 20 and 30°C. It is concluded that photoperiod and temperature are involved in the regulation of gonadal development and a long photoperiod within a suitable range of water temperatures is required for continuity of reproductive activities.

Isolation and characterization of DMRT1 and its putative regulatory region in the protogynous wrasse, Halichoeres tenuispinis

A full-length cDNA of doublesex and mab-3-related transcription factor 1 gene (DMRT1) from wrasse testis was isolated by cDNA library screening. Wrasse DMRT1 was 3116 bp in size and contained the DM domain, with a zinc finger DNA-binding motif, and the male-specific motif. Northern blot analysis identified a 3.2-kb transcript approximately equal in size to the DMRT1 nucleotide sequence detected in the testis, but not in the ovary, confirming that this sequence is male-specific in protogynous wrasse. Southern blot analysis suggested that the wrasse genome contains two copies of the DMRT1 gene. The ORF consisted of five exons and four introns with conserved donor-acceptor splice sites at all exon-intron junctions. The 5-flanking region of the wrasse DMRT1 gene was isolated by DNA walking, and putative regulatory sites were identified by searching data bases. The 5-flanking region was divided into 9 elements, then 17 DMRT-luciferase chimeric plasmids were constructed. By transient transfection into Cos-1 and TM4 cells, distal element I which contains GATA-binding sites and proximal element B containing the sex-determining region on Y chromosome gene (SRY) binding site were revealed to have an important role in transcriptional regulation of the wrasse DMRT1 when an enhancer sequence was provided.

Hyperosmotic shock adaptation by cortisol involves upregulation of branchial osmotic stress transcription factor 1 gene expression in Mozambique Tilapia.

The Mozambique tilapia (Oreochromis mossambicus) is a euryhaline species that does not survive direct seawater exposure. Cortisol is involved in re-establishing electrolyte homeostasis in seawater and is thought to play a role in allowing tilapia to cope with abrupt seawater exposure, but the mechanism(s) are far from clear. Recently, osmotic stress transcription factor 1 (OSTF1) was identified as a key signaling molecule involved in hyperosmotic stress adaptation in tilapia. Consequently, we tested the hypothesis that upregulation of OSTF1 expression by cortisol is a key response for hyperosmotic stress adaptation in tilapia. Fish were exposed to different salinities over a 24h period, while a major electrolyte disturbance and mortality was observed only with full-strength seawater exposure. Therefore, we administered cocoa butter implants of cortisol (50mg/kg) intraperitoneally to tilapia maintained in fresh water and after three days exposed these fish to full-strength seawater. There was 50% mortality in the control fish upon seawater exposure, but this was abolished by cortisol treatment. Abrupt seawater exposure did not affect plasma cortisol levels, while, as expected, exogenous administration of this steroid elevated plasma cortisol levels both in fresh water and seawater. Cortisol treatment significantly induced OSTF1 gene expression in fresh water tilapia, and also enhanced further the seawater-induced OSTF1 mRNA abundance. Plasma osmolality decreased, while gill Na(+)/K(+)-ATPase activity was suppressed in the cortisol group in seawater compared to the sham group. This corresponded with a significant reduction in gill ionocyte size and Na(+)/K(+)-ATPase activity and protein expression after seawater exposure. Cortisol did not modify liver metabolism, but significantly suppressed gill metabolic capacity in seawater. Overall, cortisol adapts tilapia to a hyperosmotic shock associated with abrupt seawater exposure. This involves upregulation of OSTF1 gene expression and a concomitant suppression of branchial metabolism in tilapia.

Regulation of sperm flagellar motility activation and chemotaxis caused by egg-derived substance(s) in sea cucumber.

The sea cucumber Holothuria atra is a broadcast spawner. Among broadcast spawners, fertilization occurs by means of an egg-derived substance(s) that induces sperm flagellar motility activation and chemotaxis. Holothuria atra sperm were quiescent in seawater, but exhibited flagellar motility activation near eggs with chorion (intact eggs). In addition, they moved in a helical motion toward intact eggs as well as a capillary filled with the water layer of the egg extracts, suggesting that an egg-derived compound(s) causes motility activation and chemotaxis. Furthermore, demembranated sperm flagella were reactivated in high pH (> 7.8) solution without cAMP, and a phosphorylation assay using (gamma-32P)ATP showed that axonemal protein phosphorylation and dephosphorylation also occurred in a pH-dependent manner. These results suggest that the activation of sperm motility in holothurians is controlled by pH-sensitive changes in axonemal protein phosphorylation. Ca2+ concentration affected the swimming trajectory of demembranated sperm, indicating that Ca2+-binding proteins present at the flagella may be associated with regulation of flagellar waveform. Moreover, the phosphorylation states of several axonemal proteins were Ca2+-sensitive, indicating that Ca2+ impacts both kinase and phosphatase activities. In addition, in vivo sperm protein phosphorylation occurred after treatment with a water-soluble egg extract. Our results suggest that one or more egg-derived compounds activate motility and subsequent chemotactic behavior via Ca2+-sensitive flagellar protein phosphorylation.

Photic and circadian regulation of melatonin production in the Mozambique tilapia Oreochromis mossambicus

Diverse circadian systems related to phylogeny and ecological adaptive strategies are proposed in teleosts. Recently, retinal photoreception was reported to be important for the circadian pacemaking activities of the Nile tilapia Oreochromis niloticus. We aimed to confirm the photic and circadian responsiveness of its close relative-the Mozambique tilapia O. mossambicus. Melatonin production in cannulated or ophthalmectomized fish and its secretion from cultured pineal glands were examined under several light regimes. Melatonin production in the cannulated tilapias was measured at 3-h intervals; it fluctuated daily, with a nocturnal increase and a diurnal decrease. Exposing the cannulated fish to several light intensities (1500-0.1 lx) and to natural light (0.1 and 0.3 lx) suppressed melatonin levels within 30 min. Static pineal gland culture under light-dark and reverse light-dark cycles revealed that melatonin synthesis increased during the dark periods. Rhythmic melatonin synthesis disappeared on pineal gland culture under constant dark and light conditions. After ophthalmectomy, plasma melatonin levels did not vary with light-dark cycles. These results suggest that (1) Mozambique tilapias possess strong photic responsiveness, (2) their pineal glands are sensitive to light but lack circadian pacemaker activity, and (3) they require lateral eyes for rhythmic melatonin secretion from the pineal gland.

Tropical monsoon environments and the reproductive cycle of the orange-spotted spinefoot Siganus guttatus

Abstract Negligible fluctuation in water temperature and photoperiod may cause fish species in tropical environments to utilize additional environmental factors to initiate reproductive activities during suitable periods of the year. We examined periodic changes in ovarian development of orange-spotted spinefoot (Siganus guttatus) in coral reefs off Karimunjawa Archipelago, Indonesia, and compared them with changes in regional environmental factors. The gonadosomatic index of this species increased initially during March–May and again during September–November. Concomitant with the increase, yolk-laden oocytes were observed (histologically) in ovaries, suggesting that the ovary develops twice a year. Initiation of reproductive activities was consistent with transition periods between rainy and dry seasons, suggesting that environmental cues linked to tropical monsoons influence reproductive activity. This suggestion is inconsistent with previous findings for the same species, but in higher latitudes, that the initiation of reproductive activity was influenced by increases in photoperiod and water temperature. It was observed that spinefoot exhibit plasticity in the use of environmental factors within habitats. Synchronized spawning occurred twice during the reproductive season, and was repeated between the new and first quarter of the lunar cycle. This periodicity was consistent with the spawning rhythm in other regions, suggesting that this species may perceive cues from the moon to synchronize spawning.

Diurnal expression patterns of neurohypophysial hormone genes in the brain of the threespot wrasse Halichoeres trimaculatus

The aim of this study was to determine the involvement of neurohypophysial hormones in the diurnal patterns of the threespot wrasse Halichoeres trimaculatus, which is common in coral reefs and exhibits daily behavioral periodicity. Prohormone cDNAs of the neurohypophysial peptides, arginine vasotocin (AVT) and isotocin (IT), were cloned by 3- and 5-rapid amplification of cDNA ends (RACE). The distribution and expression patterns of pro-AVT and -IT mRNAs in the brain were determined using reverse-transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR, respectively. The respective full-length cDNAs of pro-AVT and -IT were 945 and 755 bp in length, respectively. The deduced amino acid sequences for pro-AVT and pro-IT were 154 and 156 residues in length, respectively. Both pro-peptides contained a signal sequence followed by the respective hormones and neurophysin connected by a Gly-Lys-Arg bridge. Pro-AVT mRNA was detected only in the hypothalamus area, while pro-IT mRNA in the whole part of the brain. The relative abundance of pro-AVT and -IT mRNA varied according to time of day; it was significantly greater at 12:00 h than at 24:00 h. Following intraperitoneal administration of melatonin, pro-AVT mRNA abundance in the brain decreased, while pro-IT mRNA abundance remained unchanged. These results demonstrate that daily fluctuations of pro-AVT and pro-IT levels in the brain of threespot wrasse are differentially regulated.