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To clarify the mechanism of tetrodotoxin (TTX) accumulation in pufferfish, we compared the toxicity of two sets of wild juvenile “komonfugu” Takifugu flavipterus. The first set was sampled from Onisawa Fishing Port (FP) located in Okirai Bay, the Pacific Coast of Sanriku, Tohoku Area, Northern Japan. The second set was collected from the Onisawa FP and reared in an outdoor laboratory tank supplied with different seawater (Yoshihama Bay). The fish were sampled regularly and on the same days. The amount of TTX (mouse unit (MU)/fish) in the fish at Onisawa FP increased until 20 days and thereafter it did not change, while the amount of TTX in the fish in the laboratory tank remained low, and the TTX concentration (MU/g fish) decreased. Next, we compared the toxicity of wild juvenile T. flavipterus collected from Okirai Bay (Onisawa FP and Okirai FP) and Yoshihama Bay (Yoshihama FP). Large differences in TTX levels were observed among the fish from the three FPs. The amounts and concentrations of TTX in the fish at Onisawa FP were higher than those in the fish from the other two FPs. These results indicate that a large variation in toxic activity exists in the juvenile T. flavipterus in the bay of the Sanriku Coast.

We investigated Gemlik Bay of the Marmara Sea with an extensive high-resolution shallow seismic survey to locate and map active traces of the southern strand of the North Anatolian Fault (NAF). The southern strand splits from the main trace of the North Anatolian Fault about 80 km southeast of Izmit and extends as a continuous trace to the west for about 140 km to Gemlik Bay where it enters the Sea of Marmara. The seismic profiles reveal many faults that offset the Quaternary deposits. We interpret that four major fault zones play a significant role in shaping Gemlik Bay, the Gemlik, Kocadere–Mudanya, Gencali and Armutlu Faults, and propose that these faults are responsible for the formation of two prominent morphological features in the bay, namely Burgaz Depression and Gemlik Rise which are a pull-apart basin and a push-up structure, respectively. Closely spaced profiles to interpret the fault patterns and complex kinematics, therefore, are shown to be necessary in such areas. Besides active fault traces along the profiles, we also observed mass movements along the southern shore, offshore Mudanya–Zeytinbagi and Kursunlu in the study area. Although no destructive earthquakes were experienced in the region recently the mass movements observed on the seafloor indicate remarkable past activity along the southern strand of the North Anatolian Fault which is overshadowed by the northern strand in 20th century from seismicity point of view.

The existence of a food-entrainable circadian oscillator (FEO) was examined in goldfish Carassius auratus. Single goldfish were exposed to light–dark (LD) 12:12 (lights on 06:00–18:00 h) and fed at 12:00 h (phase I). The photoperiod regime was shifted to constant light (LL) (phase II). The fish were then fed at 06:00 or at 18:00 h (phase III), and all fish were fasted (phase IV). In both groups, three out of eight fish exhibited food-anticipatory activity (FAA) in phases II and III, the free-running period (τ) of locomotor activity was approximately 24.0 h, and circadian rhythms of locomotor activity started to free-run from the previous FAA in phase IV, indicating the existence of an FEO. Next, single goldfish were exposed to LD 12:12 and were fed twice daily at 12:00 and 00:00 h (phase I). The photoperiod regime was shifted to LL (phase II), and the fish were fasted (phase III). In phase II, FAA was observed in three out of six fish for both feeding times. In phase III, a circadian rhythm of locomotor activity was observed in five fish, and circadian rhythms started to free-run from the previous feeding time (either 12:00 or 00:00 h) in three fish. These results suggest that individual goldfish can remember two daily feeding times.

Immunohistochemical localization of orexin/hypocretin in the brain of a pleuronectiform fish, the barfin flounder Verasper moseri was examined as the first step in unraveling the possible function of the hormone in the brain. Orexin-A-like immunoreactive (ir) cell bodies were found to be located in the nucleus posterioris periventricularis (NPPv) of the hypothalamus, and orexin-A-like-ir fibers were detected not only in the hypothalamus but also extensively throughout the brain. The orexin-A-like-ir cell bodies did not project their fibers to the pituitary gland. Since melaninconcentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH) are suggested to regulate food intake in addition to orexin/hypocretin in the teleost fish, it was examined whether neural connections exist between orexin neurons and the MCH and α-MSH neurons in the barfin flounder brain by using double-staining immunohistochemistry. Some orexin-A-like-ir fibers were in close contact with the MCH-ir and α-MSH-ir cell bodies in the hypothalamus. Moreover, a few MCH-ir and α-MSH-ir fibers were in close contact with the orexin-A-like-ir cell bodies in the hypothalamus. These results suggest that reciprocal connections exist between the orexin and MCH neurons and between the orexin and α-MSH neurons in the brain of the barfin flounder.

The role of gonadotropin (GTH) in the reproduction of the Japanese flounder, Paralichthys olivaceus, was studied by assessing the changes in the apparent activity of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the pituitary gland during gonadal maturation by immunohistochemical analyses. Corresponding changes in plasma levels of testosterone (T), estradiol-17beta (E(2)), and 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) were also studied. Reared fish at the early spawning to termination stages were sampled from May to August and wild fish at the previtellogenic to termination stages were caught at 3- to 4-week intervals between April and September offshore from the northern mainland of Japan by gill nets. The gonadosomatic index of the reared fish decreased from the early spawning stage to the termination stage, while that of the wild fish increased significantly from the previtellogenic stage to the early spawning stage and decreased thereafter. In the reared fish, the immunostaining intensities of FSH and LH were high during the spawning period, accompanied by high plasma levels of T, E(2), and DHP. In the wild fish, the immunostaining intensities of FSH and LH were low during the previtellogenic stage but increased during the maturing and spawning stages. These results indicate that both FSH and LH are likely associated with oocyte maturation in the Japanese flounder.

To clarify the possible roles of gonadotropin-releasing hormone (GnRH) in the reproduction of Japanese flounder Paralichthys olivaceus, localization of salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and sea bream GnRH (sbGnRH) immunoreactive (ir) cell bodies and fibers in the brain and pituitary were examined together with follicle stimulating hormone (FSH) and luteinizing hormone (LH)-ir cells in the pituitary by immunohistochemistry. sGnRH-ir cell bodies were localized in the ventromedial part of the rostral olfactory bulb and cGnRH-II-ir cell bodies were restricted to the midbrain tegmentum, while sbGnRH-ir cell bodies were evident in the preoptic area. sGnRH-ir fibers were distributed throughout the brain, especially abundant in the forebrain. cGnRH-II-ir fibers were also scattered in many areas of the brain with abundance in the midbrain, but sbGnRH-ir fibers were observed in the preoptic-hypothalamic area and innervated the pituitary. In the pituitary, neither sGnRH-ir fibers nor cGnRH-II-ir fibers were found, but sbGnRH-ir fibers were profuse in the neurohypophysis and invaded the proximal pars distalis, targeting FSH and LH cells. These results suggest that three GnRH systems can play different physiological roles in the brain of Japanese flounder. Among them, sbGnRH is considered to be involved in reproduction by stimulating gonadotropin secretion, while sGnRH and cGnRH-II can function as a neurotransmitter and/or neuromodulator within the brain in this species.

Orexin/hypocretin is a neuropeptide that is involved in the regulation of feeding behavior and the sleep-wakefulness cycle in mammals. Melanin-concentrating hormone (MCH) is believed to be another candidate involved in food intake in teleost fish as well. Thus, it is interesting to examine whether neural connections exist between the neurons producing these two hormones. We first examined the localization of orexin-like immunoreactivity (orexin-LI) in the brain of the medaka Oryzias latipes by using immunohistochemistry. We further examined the interaction between the orexin and MCH neurons in the medaka brain by performing double-staining immunohistochemistry. Orexin-LI cell bodies were located in the nucleus posterioris periventricularis (NPPv) of the hypothalamus, and orexin-LI fibers were detected not only in the hypothalamus but also extensively throughout the brain. Some orexin-LI fibers were in close contact with the MCH-immunoreactive (ir) cell bodies in the hypothalamus, as revealed by double-staining immunohistochemistry. Moreover, a few MCH-ir fibers were in close contact with the orexin-LI cell bodies. These results suggest that in the medaka brain, orexin performs various functions, including neuromodulation, and that neural connections exist between the orexin and MCH neurons.

The self-feeding activity of the barfin flounder ( Verasper moseri ) was examined under natural photoperiod and temperature. The experiment, carried out over 12 weeks from September to December, involved tagged fish (mean body weight: 371 g) reared in three 1000-l tanks (3 or 5 fish per tank) with a self-feeder device. The sensor comprised a switch and trigger string with a bead. Self-feeding activity was recorded in all tanks from day 1. The fish fed mostly at night. Strong positive correlations were observed between the number of daily feeder activations and water temperature/photoperiod, and all fish grew during the experiment. These results demonstrate that barfin flounder can operate self-feeding systems and it is suggested that self-feeding activity is influenced by photoperiod and water temperature.

Neuropeptides with the Arg-Phe-amide motif at their C termini (RFamide peptides) were identified in the brains of several vertebrates, and shown to have important physiological roles in neuroendocrine, behavioral, sensory, and autonomic functions. The present study identified RFamide peptides, which are teleost prolactin-releasing peptide (PrRP) homologs, in the sea lamprey, Petromyzon marinus and characterized their effect on the release of pituitary hormones in vitro. Two RFamide peptides (RFa-A and RFa-B) were isolated from an acid extract of sea lamprey brain, including hypothalamus by Sep-Pak C18 cartridge, affinity chromatography using anti-salmon PrRP serum, and reverse-phase HPLC on an ODS-120T column. Amino acid (aa) sequences and mass spectrometric analyses revealed that RFa-A and RFa-B consist of 25 and 20 aa, respectively, and have 75% sequence identity within the C-terminal 20 aa. The RFa-B cDNA encoding a preprohormone of 142 aa was cloned from the lamprey brain, and the deduced aa sequence from positions 48-67 was identical to the sequence of RFa-B. However, the preprohormone does not include an aa sequence similar to the RFa-A sequence. Cell bodies, which were immunoreactive to anti-salmon PrRP serum, were located in the periventricular arcuate nucleus, ventral part of the hypothalamus, and immunoreactive fibers were abundant from the hypothalamus to the brain. A small number of immunoreactive fibers were detected in the dorsal half of the rostral pars distalis of the pituitary, close to the GH-producing cells. In addition, anti-salmon PrRP immunoreactivities were observed in the pars intermedia, corresponding to melanotropin cells. Likewise, signal of RFa-B mRNA was detected not only in the brain but also in the pars intermedia. The synthetic RFa-A and -B inhibited GH mRNA expression in a dose-dependent fashion in vitro, which is comparable to the inhibitory effect of teleost PrRP on GH release. Both RFa-A and -B also inhibited the expression of proopiomelanotropin mRNA, but no effects were observed in the expression of proopiocortin and gonadotropin beta mRNAs. The results indicate that RFamide peptides, which are teleost PrRP homologs, are present in the hypothalamus and pituitary of sea lamprey, and may be physiologically involved in the inhibition of GH and melanotropin release in the sea lamprey pituitary.

Self-feeding of grouped ayu Plecoglossus altivelis altivelis was examined under an artificial light-dark (LD) cycle and natural day length using a trigger with a photosensitive sensor. In Experiment 1, approximately 15 juvenile fish (mean body weight, 0.6 g) were kept in 60-L glass tanks (four tanks) under LD 12∶12 (lighting period 06∶00–18∶00 hours) and self-feeding and locomotor activities were simultaneously recorded. Self-feeding was detected within one day and feeding activities were strongly synchronized to the LD cycle, with almost strictly diurnal feeding (98.2%). Although locomotor activities in the upper part of the tank were also greatest during the light phase (76.8%), the activity profiles were different from those of self-feeding, suggesting the validity of the photosensitive sensor. In Experiment 2,25 fish (mean body weight, 15 g) were kept in outdoor 1000-L tanks (four tanks) under natural day length and water temperatures in May, and self-feeding was recorded from mid-August. Stable self-feeding was observed from early September in all the tanks. Nearly all food demands were diurnal, and usually crepuscular. Somatic growth was seen in all tanks. These results demonstrate that the self-feeding system using a photosensitive sensor is applicable for grouped juvenile ayu as small as 0.6 g and adult fish reared under natural conditions.

In order to develop a highly efficient method for mass production of triploid Pacific abalone Haliotis discus hannai, caffeine treatment that is safe and inexpensive was optimized. To suppress the first meiotic division, fertilized eggs were exposed to either a 10- or 15-mM caffeine solution for 24 min beginning at 12 min after fertilization. In most treated batches, the rates of cleaved eggs showed no significant difference from the control batches. However, in most treated batches, the rates of occurrence of normal larvae and the survival rates of the early juveniles were significantly lower than those of the controls. The triploid rates at 6 days to 11 months after settlement in all the treated batches were extremely high (91–100%). There was no significant difference in the mean triploid rates between 10- and 15-mM caffeine treatments. These results suggest that both treatment conditions were conducive to triploid abalone production. One live 2n/3n mosaic specimen was found in the treated batches. However, since the frequency of mosaic was extremely low, the mosaicism would probably not have an adverse effect on the stable production of triploid abalones.

Profiles of alpha-melanocyte-stimulating hormone (alpha-MSH) in the Japanese flounder were examined by a newly developed time-resolved fluoroimmunoassay (TR-FIA) and immunohistochemistry. A TR-FIA for alpha-MSH was newly developed, and its levels in the pituitary gland and plasma of Japanese flounder reared in a white or black tank for 5 months were compared. A competitive assay using two antibodies was performed among secondary antibodies in the solid phase, alpha-MSH antibodies, samples, and europium-labeled Des-Ac-alpha-MSH. The sensitivity of the assay, defined as twice the standard deviation at a zero dose, was 0.98 ng/ml (49 pg/well). The intra- and interassay coefficients of variation of the assay were 8.8% (n=8) and 17.3% (n=5), respectively, at about 50% binding. Cross-reactivities of Des-Ac-alpha-MSH and Di-Ac-alpha-MSH were about 100%. Cross-reactivities of adrenocorticotropic hormone, salmon gonadotropin-releasing hormone (sGnRH), and chicken GnRH-II were less than 0.2%, and that of melanin-concentrating hormone was less than 2.0% at 50% binding. Displacement curves of serially twofold-diluted hypothalamus extract, pituitary gland extract, and plasma extract of Japanese flounder with the assay buffer were parallel to the alpha-MSH standard curve. Moreover, displacement curves of serially twofold-diluted hypothalamus and/or pituitary gland extract of masu salmon, goldfish, red seabream, Japanese eel, tiger puffer, and barfin flounder with the assay buffer were also parallel to the alpha-MSH standard. In Japanese flounder, total immunoreactive (ir)-alpha-MSH levels in the pituitary gland were lower in the black tank, whereas those in the plasma tended to be higher in the black tank, suggesting that the synthesis and release of alpha-MSH are higher in the black tank. alpha-MSH-ir cells were detected in the pars intermedia and a small part of the pars distalis of the pituitary gland. alpha-MSH-ir cell bodies were located in the basal hypothalamus and alpha-MSH-ir fibers were distributed not only in the hypothalamus but also in the telencephalon, midbrain, cerebellum, and medulla oblongata, suggesting that alpha-MSH functions as a neuromodulator in the brain.

The telomere sequence type (TTAGGG)n is known to be distributed in various phyla in the Animalia and in Mastigophora (Protista). However, the telomere type of Porifera (sponges), a phylum comprising the lowest multicellular animals, has not been reported. In this study, we examined the three sponge species Leucetta chagosensis, Halichondria japonica, and Halichondria panicea for the presence of the telomere type (TTAGGG)n by fluorescence in situ hybridization (FISH). The oligonucleotide probe (TTAGGG)7 clearly displayed signals on the interphase nuclei of all three sponges. In contrast, the (TTAGG)7 probe, which has one base fewer than (TTAGGG)7, did not display the signals. These results suggest that the telomeres of the three sponges consist of (TTAGGG)n, which is identical to the sequence type found in many higher multicellular animals and in Mastigophora. Additionally, this is the first study to reveal a telomere sequence type for Porifera. Moreover, these results suggest that Porifera are phylogenetically related to Mastigophora, and supports the general theory that Porifera evolved from Mastigophora. Further, this study strongly suggests that the origin of the (TTAGGG)n telomere sequence is to be found in a common ancestor of either the Bilateria and Porifera, or the Protista.

Immunohistochemical localization and ontogenic development of prolactin-releasing peptide (PrRP) in the brain of the ovoviviparous fish species Poecilia reticulata (guppy) were examined to gain a better understanding of this hormone in teleost fish. In adult guppies, PrRP-immunoreactive (ir) cell bodies were detected in the posterior part of the hypothalamus. In the pituitary, a small number of PrRP-ir fibers were observed adjacent to the prolactin cells, whereas numerous PrRP-ir fibers were detected not only in the hypothalamus but also widely throughout the brain. PrRP-ir cell bodies and prolactin cells were already detected on the birth day in the hypothalamus and pituitary, respectively. The number of PrRP-ir fibers in the brain increased as the fish developed. These results suggest that PrRP is involved in neuromodulation in the brain and that PrRP plays some physiological roles in the early development of the guppy.

To elucidate the role of gonadotropin-releasing hormone (GnRH) in gonadal maturation in wild female Japanese flounder Paralichthys olivaceus, we monitored changes in the levels of seabream GnRH (sbGnRH) in the olfactory bulb, telencephalon, hypothalamus, and pituitary during ovarian development together with changes in plasma levels of testosterone (T), estradiol-17β (E2), and 17α, 20β-dihydroxy-4-pregnen-3-one (DHP). Fish were caught offshore of the northern mainland of Japan in the Pacific Ocean at 3- to 4-week intervals between April and September by gill net. The netted fish were categorized into six groups based on ovarian stages: previtellogenic (April–early May), early yolk (April–late May), late yolk (late May–June), early spawning (June–August), late spawning (September), and termination (September) stages. The gonadosomatic index significantly increased from the previtellogenic to early spawning stages and decreased thereafter. In the olfactory bulb, no significant differences were observed in sbGnRH levels among the developmental stages. In contrast, sbGnRH levels in the telencephalon and hypothalamus were very high in the previtellogenic stage, lower in the early spawning stage, and relatively high in latter stages. sbGnRH levels in the pituitary were high in the previtellogenic stage and low in the early spawning stage. In addition, the relatively high levels of pituitary sbGnRH were found together with high plasma T, E2, and DHP levels in fish in the late yolk stage. These results indicate that sbGnRH in the telencephalon, hypothalamus, and pituitary is involved in ovarian maturation and that sbGnRH may play an important role in the initiation of ovarian recrudescence in wild Japanese flounder.

The self-feeding rhythms of ayu Plecoglossus altivelis altivelis were examined. Individual ayu (mean body weight 40 g) were held in 60-L glass tanks equipped with self-feeders. Six of 14 fish learned self-feeding during the experiment. Under two different light-dark (LD) conditions (16 h:8 h and 8 h:16h LD), self-feeding was synchronized to the LD cycle, and feeding occurred almost exclusively during the light phase. During exposure to constant light (LL), circadian feeding rhythms were observed. These results indicate self-feeding rhythms in ayu are restricted to the light phase under LD conditions and are controlled by the circadian clock under LL conditions.

Wild a dult maturing and immature female Japanese flounder Paralichthys olivaceus were collected in June 2004 and January 2005, respectively, to clarify a possible role of gonadotropin-releasing hormones (GnRHs) in reproduction. Levels of salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and sea bream GnRH (sbGnRH) in the brain and pituitary were examined by time-resolved fluoroimmunoassay. Three forms of GnRHs were detected in the discrete brain at various levels. In the pituitary of both maturing and immature fish, sbGnRH was abundant together with a pronounced amount of sGnRH, whereas cGnRH-II was almost below the detectable limit. In maturing fish, levels of sbGnRH were high in the telencephalon, hypothalamus and pituitary, while levels of sbGnRH of immature fish were very low in these regions. These results indicate that sbGnRH is mainly responsible for gonadotropin secretion, and that sb GnRH in the anterior part of the brain is associated with gonadal maturation in the Japanese flounder.

A pleuronectiform fish, the barfin flounder Verasper moseri, reared in a white tank had a smaller ratio of pigmented area of the skin on non-eyed side, grew faster, and had greater melanin-concentrating hormone (MCH)-immunoreactive cell bodies and MCH gene expression in the brain than in the black tank, indicating that synthesis and release of MCH are higher in fish from a white tank. In the present study, a time-resolved fluoroimmunoassay for MCH was developed. MCH levels were assessed in the brain, pituitary gland, and plasma of barfin flounders reared in a white or black tank. A competitive assay using two antibodies was performed among secondary antibodies in the solid phase, MCH antibodies, samples, and europium-labeled MCH. Displacement curves of serially diluted extracts (brain, pituitary gland, and plasma) of the barfin flounder paralleled that of the MCH standard. MCH levels in the brain and plasma were higher in fish reared in the white tank for 5 months than in the black tank. These results suggest that synthesis and secretion of MCH are enhanced with the white background and that MCH is involved in both somatic growth and the skin pigmentation in the barfin flounder.

Effects of feeding time on approaching behavior to food odor in goldfish Carassius auratus were examined using the Y-maze flow-through system to test whether the behavior differed according to feeding time and experimental time. Fish were fed at 08.00, 12.00 or 16.00 hours once a day or three times (08.00, 12.00 and 16.00 hours) a day for at least 3 weeks under a natural photoperiod, and at 22±2°C, preference reactions to food odor were tested at 08.00, 10.00, 12.00, 14.00 and 16.00 hours. The fish fed once a day showed significant positive approaching behavior only at the time when they were normally fed. The fish fed three times a day showed no significant positive approaching behavior. These results suggest that golfish can remember when food is available and/ or that there exists a mechanism to inhibit approaching behavior when food is not available.

A pleuronectiform fish, the barfin flounder Verasper moseri, expresses three gonadotropin-releasing hormone (GnRH) forms in the brain: salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and seabream GnRH (sbGnRH). To clarify the effects of photoperiod on GnRH systems, changes in brain and pituitary GnRH peptide levels were examined using time-resolved fluoroimmunoassays. In experiment 1, 5-month-old male barfin flounder (mean total length 9.0 cm, body weight 11.0 g) were divided into short (8:16 h light : dark [L:D] cycle; lights on 08.00–16.00 hours) and long photoperiod (16:8 h L:D cycle; lights on 04.00–20.00 hours) groups in mid September and maintained until November under natural water temperature (19.3–15.2°C). Brain sGnRH concentrations were significantly higher in the 16:8 h L:D group than in the 8:16 h L:D group, whereas no significant differences were observed in total length, body weight, plasma testosterone concentration, brain cGnRH-II concentration and pituitary sbGnRH content. In experiment 2, 7-month-old male barfin flounder (mean total length 16.5 cm, body weight 76.8 g) were divided into short and long photoperiod groups in mid December and maintained until February under natural water temperature (12.5–6.6°C). Total length, body weight and condition factor were significantly greater in the 16:8 h L:D group than in the 8:16 h L:D group, whereas no significant differences were observed in plasma testosterone concentration and GnRH levels in the brain and pituitary. These results indicate that levels of sGnRH in barfin flounder are influenced by photoperiodic treatment dependent on water temperature and/or body size.

Effects of gonadotropin-releasing hormone (GnRH) on thyroxine (T4) release in vivo and in vitro were studied in barfin flounder Verasper moseri, masu salmon Oncorhynchus masou and goldfish Carassius auratus. Seabream GnRH (sbGnRH) at a dose of 200 ng/50 g body weight (BW) significantly increased plasma T4 levels 1 h after the in vivo injection in the barfin flounder, but thereafter the levels normalized. Salmon GnRH (sGnRH) significantly increased plasma T4 levels l h after the injection with a significant return to initial levels in male masu salmon and male goldfish. In contrast, sGnRH and cGnRH-II in barfin flounder, and cGnRH-II in male masu salmon and male goldfish were not effective in stimulating T4 release. To clarify direct involvement of GnRH in T4 release, dissected lower jaw including scattered thyroid follicles was incubated with sbGnRH (1 µg/well) in barfin flounder, and with two doses (0.1 and 1 µg/well) of sGnRH in masu salmon and goldfish in vitro. T4 concentrations of control were stable during 24 h. Incubation of lower jaw with high dose (1 µg/well) of GnRH significantly (P

The pleuronectid barfin flounder (Verasper moseri) expresses three forms of gonadotropin-releasing hormone (GnRH) in the brain. To clarify the physiological roles of the respective forms during testicular maturation, changes in brain GnRH mRNA levels and pituitary GnRH peptide levels were examined by real-time quantitative PCR and time-resolved fluoroimmunoassay, respectively. Fish hatched in April 2000. The gonadosomatic index remained low until October 2001 and then rapidly increased in January 2002. Fish continued to grow from hatching through testicular maturation. Fish spermiated in March 2002. The amount of seabream GnRH (sbGnRH) mRNA per brain significantly increased in January 2002 and remained at high levels in March 2002. The amounts of salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II) mRNA per brain did not show significant changes during the experimental periods. Pituitary sbGnRH peptide content significantly increased in March 2002. Pituitary sGnRH peptide and cGnRH-II peptide contents were extremely low compared to sbGnRH peptide levels and showed no significant changes during the experiment. These results indicate that sbGnRH is involved in the testicular maturation of barfin flounder.

Variety in histochemical characteristics of the olfactory receptor cells (ORC) was examined by immunohistochemistry for protein gene product 9.5 (PGP9.5) and calretinin, and by lectin histochemistry with Phaseolus vulgaris leucoagglutinin (PHA-L) in the olfactory epithelium (OE) of the barfin flounder (Verasper moseri). PGP 9.5 immunoreactivity was observed in the ORC situated in the upper three fourths of the OE. Calretinin immunoreactivity was observed in the ORC which seemed to be immunonegative for PGP 9.5. These cells were located in the upper two thirds of the OE. PHA-L staining was observed in small subsets of the ORC. PGP 9.5 and calretinin immunoreactivities and PHA-L staining were also observed in the crypt cells unique to the fish OE. These findings suggest the different properties of olfactory perception among fish ORC.

Melanin-concentrating hormone (MCH) was first discovered in the pituitary of chum salmon because of its role in the regulation of skin pallor. Later, it was found that MCH could also play a role as a central neurotransmitter or neuromodulator in the brain. However, knowledge of the function of MCH in fish has been restricted to certain fish species. Therefore, in the present study, the immunocytochemical localization and ontogenic development of MCH in the brain of a pleuronectiform fish, the barfin flounder Verasper moseri, were examined to obtain a better understanding of this hormone. In adult barfin flounder, MCH-immunoreactive (ir) neuronal somata were most prevalent in the magnocellular neurons of the nucleus tuberis lateralis (NLT), which project to the pituitary. In the pituitary, MCH-ir fibers were distributed in the neurohypophysial tissues within the pars intermedia and, to a lesser extent, into the pars distalis. MCH-ir neuronal somata were also present in dorsally projecting parvocellular neurons, located more posteriorly in the area above the lateral ventricular recess (LVR). LVR-MCH neurons did not seem to project to the pituitary. In the brain, MCH-ir fibers were detected not only in the hypothalamus but also in areas such as the optic tectum and thalamus. MCH-ir neuronal somata and fibers were not detected on the day of hatching. MCH-ir neuronal somata and fibers were first detected in the hypothalamus and the pituitary, respectively, 7 days after hatching. Subsequently, MCH-ir neuronal somata were observed in the NLT and in the area above the LVR 14 days after hatching. The distribution of MCH-ir neuronal somata and fibers showed a pattern similar to that in the adult fish 35-42 days after hatching. These results indicate that MCH neurons were located in the NLT and in the area above the LVR and that NLT-MCH neurons project to the pituitary. MCH neurons were first detected 7 days after hatching, suggesting that MCH plays some physiological role in the early development of barfin flounder.

Multiple forms of the gonadotropin-releasing hormone (GnRH) exist in teleost fish. A salmonid fish, masu salmon Oncorhynchus masou has salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). sGnRH neurons were scattered from the olfactory nerve through the ventral telencephalon (VT) and the preoptic area (POA). sGnRH but not cGnRH-II was detected in the pituitary. sGnRH mRNA levels in the VT and the POA increased during gonadal maturation, suggesting that sGnRH neurons in these areas are involved in gonadal maturation. sGnRH neurons were first detected in a cluster near the olfactory epithelium 40 days after fertilization. sGnRH neurons were not detected in the brain by the olfactory epithelia lesion, suggesting that sGnRH neurons are derived from the olfactory epithelium. A pleuronectiform fish, barfin flounder Verasper moseri has sGnRH, cGnRH-II and seabream GnRH (sbGnRH). sGnRH and cGnRH-II-immunoreactive fibers were observed throughout the brain, but not in the pituitary. sbGnRH neurons were located in the POA and sent fibers to the pituitary, indicating that sbGnRH is involved in GTH secretion. Judging from the location of neuronal somata and their projections, it is indicated that three GnRH systems exist in the barfin flounder; the TN-, the MT- and the POA-GnRH system. However, in masu salmon, clear anatomical identification of the TN- and the POA-GnRH system is difficult, because the GnRH neurons located in the ventral forebrain are consecutive and the GnRH form produced in these neurons is the same (sGnRH). Thus, it is suggested in masu salmon that sGnRH neurons are derived from the olfactory epithelium, migrate into the brain and play different roles according to the location in the brain.

During the ontogeny of masu salmon Oncorhynchus masou, neurons producing the salmon type of gonadotropin-releasing hormone (sGnRH) were first detected in the olfactory epithelium of the eyed egg and, subsequently, in the brain, suggesting a migration of these cells. Among sGnRH neurons distributed from the olfactory nerve (ON) through the preoptic area (POA), those in the ventral telencephalon (VT) and the POA are indicated to regulate gonadotropin secretion. Thus, it is of interest to know whether all the sGnRH neurons originate from the olfactory epithelium. In the present study, we examined by in situ hybridization whether sGnRH neurons are present in the VT-POA of fish, whose olfactory epithelia including sGnRH clusters were cauterized just after hatching (44 days after fertilization). Fish were sampled in June (212 days after the operation). Neurons expressing sGnRH mRNA were detected in the VT-POA as well as in the ON, ventral olfactory bulb, and transitional area between the olfactory bulb and telencephalon (which is considered to correspond to the terminal nerve ganglion) in the control group. In contrast, neurons expressing sGnRH mRNA were not detected in the VT-POA in the olfactory epithelium lesioned (OEL) group. Furthermore, pituitary sGnRH content in the OEL group was just above the detectable limit and was significantly lower than that in the corresponding control group in both sexes. These results indicate that sGnRH neurons in the VT-POA are derived from the olfactory epithelium in masu salmon, although the possibility cannot be ruled out that sGnRH neurons in the VT-POA arise from the VT-POA, but were delayed in expressing sGnRH because of the trauma of cauterization.

Testicular maturation of underyearling precocious male masu salmon (Oncorhynchus masou) is affected by photoperiod. It is accelerated by a short photoperiod (light-dark cycles of 8:16 h; LD 8:16) and delayed by a long photoperiod (LD 16:8). Circulating melatonin levels are high during the night and low during the day:the duration of the nocturnal elevation is longer under a short than under a long photoperiod, suggesting mediation of photoperiodic signals by melatonin. This study examined whether melatonin administration mimics short photoperiodic effects and whether it accelerates the testicular development of underyearling male masu salmon reared under a long photoperiod. Fish were randomly selected in June and were divided into two groups. They were reared under LD 16:8 (lights on 04:00-20:00 h) and fed pellets sprayed with melatonin (0.5 mg melatonin/kg body weight/day) or vehicle once a day at 11:00 h until October. The plasma melatonin profile of the melatonin-treated group was similar to that expected under a short photoperiod. Melatonin treatment had a stimulatory effect on the gonadosomatic index and pituitary gonadotropin (GTH) I contents. Plasma testosterone levels were significantly higher in the melatonin-treated group than in the control group in August. However, spermiation was observed in October in both groups and no significant differences were observed in GTH II contents in the pituitary in the two groups throughout the experiment. These results suggest that mimicking a short photoperiod by melatonin administration stimulated testicular development but did not completely activate the brain-pituitary-gonadal axis in precocious male masu salmon. Thus, melatonin is suggested to be one of the factors that mediates the transduction of photoperiodic information to the brain-pituitary-gonadal axis.

A fluorescence in situ hybridization (FISH) technique was used for elucidating the telomeric sequence and its chromosomal location in the Pacific abalone Haliotis discus hannai. The vertebrate telomere PNA (peptide nucleic acid) probe, the oligonucleotide (TTAGGG)7 and (TTAGG)7 probes were used for the analysis. FISH with the PNA and (TTAGGG)7 oligonucleotide probes displayed clearly defined signals on the telomeric regions of all metaphase chromosomes and interphase nuclei. On the other hand, the (TTAGG)7 probe, which has one base less than probe (TTAGGG)7, did not display clearly defined signals. These results suggest that the telomere of H. d. hannai is composed of a (TTAGGG)n sequence, which is typically found in vertebrates and other molluscs. FISH-derived signals were not observed at interstitial sites of H. d. hannai chromosomes. Hence, this species is unlikely to possess any nontelomeric sites of the telomeric sequence. The successful use of FISH in this study suggests that the FISH techn...

A purification method for tetrodotoxin (TTX), based on affinity chromatography using the TTX-binding high mol. wt substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands, was developed. This method was particularly useful for analysis of TTX in biological samples with low concentrations of TTX. The affinity gel prepared was highly specific for TTX, having no ability to bind 4-epi-TTX and anhydro-TTX as well as saxitoxin.

Catadromous female eels were administered once a week with commercial gonadotropins, DES-Na and Vitamin E for a total of six occasions, and then with the acetone-dried pituitary glands of fishes several times over a suitably short period. The administration of LH-RH analogue was also tried at the later stage of maturation. Catadromous male eels were injected with the commercial gonadotropins and V.E on more than 5 occasions. The matured female and male eels were then kept in the same tank (1.6×3.5×0.9m, water depth 0.6m) and the above-mentioned hormone treatments were continued. When they were kept at a water temperature of 18-20°C, spawning did not occur at all and even when fertilized eggs were obtained without any observable spawning behaviour the eggs did not hatch. But when the water temperature was raised to 21-22°C, spawning behaviour was observed at night and the spawning took place in the early morning. The fertilized eggs from this spawning hatched normally. The abdomen of the spawned eels swelled up once again over a considerably short duration in almost all cases, so it can be presumed that spawning can take place at least twice. It is presumed that the normal body weight of female eels ranged between 110-130% (about 120%) of that at the start of the experiment, just before spawning. Mature female eels with a body weight of more than 130% always gave rise to denatured eggs.

In puffer fish, tetrodotoxin (TTX) exists as the major toxin with chemically equilibrium analogs (4-epiTTX, 4,9-anhydroTTX) and chemically non-equilibrium analogs (deoxy analogs, 11-oxoTTX, 4-S-cysteinylTTX). There are two purposes to this study: 1) to search for the reason why TTX is the most major analog in puffer fish, even 4,9-anhydroTTX is chemically more stable, 2) to investigate whether or not chemically non-equilibrium analogs are transformed in puffer fish, because these were predicted to be biosynthetic intermediates. Pure TTX, 4-epiTTX, 4,9-anhydroTTX, and 11-oxoTTX were separately administrated to the cultured non-toxic juvenile puffer fish kusafugu, Fugu niphobles by intramuscular injection. Sixteen days after administration, TTX analogs in the whole fish were analyzed by LC-fluorescent detection and LC/MS. By the administration of TTX, 4-epiTTX, and 4,9-anhydroTTX, 34-40% of the administrated doses of the toxins were accumulated, and 4,9-anhydroTTX has become the major toxin after inter-conversion. This result indicates discrepancy from the previous ones wherein TTX was predominantly accumulated when TTXs were administrated through diets; this suggests that dietary administration might be necessary to accumulate TTX as the major toxin, and not 4,9-anhydroTTX. Transformations from TTX to deoxy analogs or 11-oxoTTX, or from 11-oxoTTX to TTX were not detected in this study.

The pleuronectid barfin flounder Verasper moseri expresses three forms of gonadotropin-releasing hormones (GnRHs), i.e., seabream GnRH (sbGnRH), salmon GnRH, and chicken GnRH-II. Among these, sbGnRH is the dominant form in the pituitary, indicating that sbGnRH regulates gonadal maturation. In order to clarify the physiological roles of sbGnRH during ovarian maturation in reared female barfin flounder, the changes in brain sbGnRH mRNA levels and pituitary sbGnRH peptide levels were examined by real-time quantitative PCR and time-resolved fluoroimmunoassay, respectively. The fish hatched in April 2002. The gonadosomatic index remained low until August 2004 and increased thereafter until April 2005 when the fish began to ovulate. The sbGnRH mRNA levels per brain increased significantly from April 2004 to April 2005. Pituitary sbGnRH peptide levels also increased significantly during this period. These results indicate that sbGnRH is involved in ovarian maturation and ovulation in the barfin flounder.

Non-toxic cultured juvenile kusafugu Fugu niphobles were fed with a diet containing highly toxic natural komonfugu Fugu poecilonotus liver until the 30th day (8.0 microg of TTX and 3.7 microg of 4,9-anhydroTTX/fish/day), and then fed with a non-toxic diet until the 240th day. During the 30-240th day, five or six fish were periodically sampled six times, and the contents of TTX and 4,9-anhydroTTX in each tissue were determined. The total TTX and 4,9-anhydroTTX accumulated in all tissues tested was not significantly changed during the experimental period, both being kept at 70% of administrated doses. However, in the liver, the TTX content accounted to be 120 microg (50% of administrated) on the 30th day, and then it gradually decreased to 50 microg until the 240th day, while 4,9-anhydroTTX content was kept at approximately 40 microg (40% of administrated) during all the experimental periods. In contrast to the liver, in the skin, TTX and 4,9-anhydroTTX were 40 and 5 microg, respectively, on the 30th day, and then gradually increased to 80 and 24 microg, respectively, until the 240th day. In the intestine, TTX and 4,9-anhydroTTX contents were kept at 25 and 12 microg, respectively, during all the experimental periods. According to these results, we assumed that a part of TTX accumulated in the liver was slowly transferred to the skin.

Effects of background color on gonadotropin-releasing hormone (GnRH) and melanin-concentrating hormone (MCH) levels in the brain of the barfin flounder Verasper moseri were monitored to investigate the interaction of GnRH and MCH in the brain. Fish were reared in white or black tanks from one month after hatching for about 7 months. MCH levels in the brain and pituitary were higher in the white tank fish. In contrast, chicken GnRH-II (cGnRH-II) levels in the brain were higher in the black tank fish. No significant differences between background colors were observed in the brain concerning salmon GnRH and seabream GnRH levels. Furthermore, six-month-old fish that had been reared in white tank were transferred to another white or black tank. Brain cGnRH-II levels were higher in black tank fish than those in white tank at 2 and 7 days after the transfer. Double-staining immunohistochemistry showed that some cGnRH-II-immunoreactive (ir) fibers were in close contact with MCH-ir cell bodies in the hypothalamus. These results indicate that background color affects not only MCH levels but also cGnRH-II levels in the brain and suggest that cGnRH-II may play a role in the regulation of MCH neural function, food intake, in the brain of the barfin flounder.

We recently identified a cDNA encoding three novel fish hypothalamic neuropeptides, having LPXRF-NH2 from the goldfish brain. In this study, to clarify the physiological functions of these three LPXRFamide peptides (gfLPXRFa-1, -2, and -3), we analysed the localisation and hypophysiotrophic activity of these peptides using sockeye salmon, Oncorhynchus nerka, in which immunoassay systems for several anterior pituitary hormones have been developed. gfLPXRFa-immunoreactive cell bodies were detected in the nucleus posterioris periventricularis of the hypothalamus and immunoreactive fibres were distributed in various brain regions and the pituitary. We also detected gfLPXRFa-immunoreactivity in the pituitary by competitive enzyme-linked immunosorbent assay combined with reversed-phase HPLC. These three gfLPXRFamide peptides stimulated the release of FSH, LH and GH, but did not affect the release of prolactin (PRL) and somatolactin (SL) from cultured pituitary cells. These results suggest that novel fish hypothalamic LPXR-Famide peptides exist in the brain and pituitary of sockeye salmon and stimulate the release of gonadotrophins and GH from the pituitary.

To test whether the affinity (Kd) and total binding capacity (Bmax) of melatonin receptors exhibit daily and circadian changes in teleost fish whose melatonin secretion is not regulated by intra-pineal clocks, we examined the changes in melatonin binding sites in the brains of underyearling masu salmon Oncorhynchus masou under artificial light-dark (LD), constant light (LL) and constant dark (DD) conditions. In Experiment 1, fish were reared under a long (LD 16:8) or short (LD 8:16) photoperiod for 69 days. Blood and brains were sampled eight times at 3 h intervals. Plasma melatonin levels were high during the dark phase and low during the light phase in both photoperiodic groups. The Bmax exhibited no daily variations. Although the Kd slightly, but significantly, changed under LD 8:16, this may be of little physiological significance. In Experiment 2, fish reared under LD 12:12 for 27 days were exposed to LL or DD from the onset of the dark phase under LD 12:12. Blood and brains were sampled 13 times at 4 h intervals for two complete 24 h cycles. Plasma melatonin levels were constantly high in the DD group and low in the LL group. No significant differences were observed in the Kd and the Bmax between the two groups, and the Kd and the Bmax exhibited no circadian variation either in the LL or DD groups. These results indicate that light conditions have little effect on melatonin binding sites in the masu salmon brain.

Proopiomelanocortin (POMC) is a common precursor of adrenocorticotropic hormone (ACTH), melanophore-stimulating hormone (MSH), and endorphin (END). In pituitary gland, POMC receives posttranslational processing by which different peptides are generated in the pars distalis (PD) and pars intermedia (PI). Recently, we cloned three subtypes of the POMC gene in pituitary gland of barfin flounder. The present study was undertaken to elucidate whether the three POMC genes are expressed in both the PD and PI of barfin flounder pituitary, and to identify peptides derived from POMCs in these lobes. We amplified the transcripts of POMC-A, -B and -C in both the PD and PI by the reverse transcription-polymerase chain reaction. In situ hybridization also detected signals for these three subtypes in the PD and PI. These results demonstrated that all three POMC genes are expressed in both the PD and PI of barfin flounder pituitary. By mass spectrometric analyses, ACTH-A, Des-acetyl (Ac)-alpha-MSH-A/B (amino acid sequence of alpha-MSH-A is identical to that of alpha-MSH-B), beta-MSH-A, corticotropin-like intermediate lobe peptide (CLIP)-A, and N-terminal peptide (N-POMC)-A were identified in the PD. Moreover, Des-Ac-alpha-MSH-A/B, alpha-MSH-A/B, beta-MSH-A and -B, N-beta-lipotropin-A, CLIP-A, N-Ac-beta-END-A(1-41) (C-terminally truncated form of N-Ac-beta-END-A), and N-POMC-A were identified in the PI. Predominant detection of POMC-A-derived peptides indicates the greatest production of POMC-A and no detection of POMC-C-derived peptides indicates the lowest production of POMC-C in both the PD and PI. ACTH-A is specifically produced in the PD, however, the occurrence of Des-Ac-alpha-MSH-A, CLIP-A, and beta-MSH-A shows that the entire POMC-A is further cleaved into small peptides as in the PI. In the PI, some peptides receive modification or truncation as shown by the occurrence of alpha-MSH-A/B and N-Ac-beta-END-A(1-41). These results show differential posttranslational processing of POMC between the PD and PI in barfin flounder pituitary.

Differentiation of the histochemical characteristics of the olfactory receptor cells (ORC) was examined by immunohistochemistry for protein gene product 9.5 (PGP 9.5) and calretinin (CR) and lectin histochemistry for Phaseolus vulgaris agglutinin-L (PHA-L) in the developing olfactory epithelium (OE) of the barfin flounder. PGP 9.5 immunoreactivity was diffuse and CR immunoreactivity was restricted at day 7, but these immunoreactivities became intense in the OE toward day 91. Crypt cells were first identified at day 56. PHA-L staining was faint at day 28, but became intense toward day 91. These findings suggest that PGP 9.5-immunopositive cells, CR-immunopositive cells, crypt cells and PHA-L-reactive cells differentiate independently in the developing OE and constitute subsets of the ORC in the OE.

Morphogenesis of the olfactory pit (OP), olfactory lamella (OL) and olfactory epithelium (OE) was examined by scanning electron and light microscopy in the barfin flounder (Verasper moseri). At day 0 after hatch, the OP was already formed. At day 14, the cellular differentiation of the OE was prominent. At day 42, the OP became a cavity by the formation of its roof. At day 56, the first OL extended remarkably and was lined with the OE on both sides. The OL increased in number with development. These findings suggest that the OE is functionally active at day 14. The formation of the OL in the OP may be initiated by the stimulus when the barfin flounder touched at the bottom of the sea.

Melanophore-stimulating hormone (MSH) has been shown to be associated with food intake in addition to body color change in teleosts. MSH is encoded by a proopiomelanocortin (POMC) gene together with endorphin (END). To assess the significance of MSH to biological activities, we determined the structure and evaluated the expression of POMC mRNA in barfin flounder (bf), Verasper moseri , a member of a group of teleosts, Pleuronectiformes. Three subtypes of POMC cDNAs (A, B, and C) were amplified from bf pituitary glands. These bfPOMCs contained segments for N -POMC, α-MSH, β-MSH, and β-END as do other teleost POMCs, while POMC-C showed remarkable variations in the segments corresponding to N -POMC and β-END. A phylogenetic tree of ray-finned fish POMCs constructed by the neighbor joining method revealed that the three POMC subtypes may have appeared as a result of duplication events occurring at least twice during the course of bf evolution. The first duplication may have generated the lineage leading to an ancestor of bfPOMC-A and -B and that leading to bfPOMC-C, and then the lineage of bfPOMC-A may have diverged from that of bfPOMC-B. All peptides flanked by processing signals excluding N -POMC-C (1–14) were identified in a single pituitary extract by mass spectrometry, and the cDNAs of three POMCs were amplified from a single pituitary by reverse transcription polymerase chain reaction. These results demonstrated that the three POMC genes are expressed in a single individual. While the bfPOMC-A gene was exclusively expressed in the pituitary, the bfPOMC-B and -C genes were expressed in non-pituitary tissues such as brain, gill, heart, spleen, liver, stomach, intestine, testis, muscle, blood, and skin in addition to the pituitary. The expression levels of the POMC-A, -B, and -C genes in pituitary neurointermediate lobe were greater in the fish reared with a black background than the fish reared with a white background, indicating that MSH derived from all of the three bfPOMC genes was associated with body color change. No difference was observed in the expression levels of bfPOMC-C in the brain in response to feeding status.

Non-toxic cultured juvenile kusafugu were fed with diet containing crystalline tetrodotoxin (TTX) for 30 days and then fed with non-toxic diet for 170 days. During this period, 5 fish were sampled and the toxicity of each tissue was determined periodically. The amount of total accumulated toxin in the fish was 90 microg, representing 50% of the administered TTX (180 microg/fish) at the 60th day. It decreased to 54 microg (30%) at the 80th day and then remained unchanged up to the 200th day. The amount of toxin in the liver amounted to 40 microg (45% of total accumulated toxin) at the 30th day and gradually decreased to 5 microg (10%) at the 200th day. The toxin amount in the skin reached the highest level with 30 microg (30%) at the 50th day and then remained unchanged during the experimental period. The testes had almost no toxicity. Although the ovaries were immature, the toxin amounts increased as the weight of the tissues increased. With administration of crystalline TTX, all kusafugu used in the experiment became toxic and retained the toxin at the level of 30% of the administered toxin for about 5 months thereafter, while being fed with non-toxic diet.

Alpha-melanocyte-stimulating hormone (alpha-MSH) is a pituitary hormone derived by post-translational processing from proopiomelanocortin and is involved in background adaptation in teleost fish. It has also been reported to suppress food intake in mammals. Here, we examined the immunocytochemical localization of alpha-MSH in the brain and pituitary of a pleuronectiform fish, the barfin flounder (Verasper moseri), as a first step in unraveling the possible function of alpha-MSH in the brain. The ontogenic development of the alpha-MSH system was also studied. In the pituitary, alpha-MSH-immunoreactive (ir) cells were preferentially detected in the pars intermedia. In the brain, alpha-MSH-ir neuronal somata were located in the nucleus tuberis lateralis of the basal hypothalamus, and alpha-MSH-ir fibers were located mainly in the telencephalon, hypothalamus, and midbrain. Alpha-MSH-ir neuronal somata did not project their axons to the pituitary. The alpha-MSH-ir neurons differed from those immunoreactive to melanin-concentrating hormone. Alpha-MSH cells in the pituitary and alpha-MSH-ir neuronal somata in the brain were first detected 1 day and 5 days after hatching, respectively. The distribution of alpha-MSH-ir cells, neuronal somata, and fibers showed a pattern similar to that in adult fish 30 days after hatching. These results indicate that the functions of alpha-MSH in the brain and pituitary are different and that alpha-MSH plays physiological roles in the early development of the barfin flounder.

A pleuronectiform fish, the barfin flounder Verasper moseri, has three molecular forms of gonadotropin-releasing hormone (GnRH) in the brain, salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and seabream GnRH (sbGnRH). To elucidate the ontogenic origin of the neurons that produce these GnRH molecules, the development of three GnRH systems was examined by in situ hybridization and immunocytochemistry. Neuronal somata that express sGnRH mRNA were detected first in the vicinity of the olfactory epithelium 21 days after hatching (Day 21), and then in the transitional area between the olfactory nerve and olfactory bulb and the terminal nerve ganglion on Day 28. cGnRH-II mRNA-expressing neuronal somata were first identified in the midbrain tegmentum near the ventricle on Day 7. cGnRH-II-immunoreactive (ir) fibers were first found in the brain on Day 7. sbGnRH mRNA-expressing neuronal somata were first detected in the preoptic area on Day 42. sbGnRH-ir fibers were localized in the preoptic area-hypothalamus, and formed a distinctive bundle of axons projecting to the pituitary on Day 70. These results indicate that three forms of GnRH neurons have separate embryonic origins in the barfin flounder as in other perciform fish such as tilapia Oreochromis niloticus and red seabream Pagrus major: sGnRH, cGnRH-II and sbGnRH neurons derive from the olfactory placode, the midbrain tegmentum near the ventricle and the preoptic area, respectively.

We have previously shown that the testicular development of underyearling male masu salmon Oncorhynchus masou reared under a long photoperiod was accelerated by oral melatonin treatment (0.5 mg melatonin/kg body weight/day), suggesting that melatonin mediates photoperiodic signaling. In this study, we further examined the effects of a disturbance in the plasma melatonin profile on gonadal development in underyearling male masu salmon by administering a higher dose of melatonin. Fish randomly selected in June were divided into two groups. They were reared under a light:dark (LD) cycle of 16:8 (lights on 04:00-20:00 hr) and fed with pellets sprayed with melatonin or vehicle twice a day at 08:30 and at 15:30 hr (7.5 mg melatonin/kg body weight/day) until October. Fish were sampled on Day 0, 25, 60, 90 and 120. The plasma melatonin levels were high in the dark phase and low in the light phase in the control group, while they were constantly high with no significant change in the melatonin-treated group. Melatonin treatment had inhibitory effects on the gonadosomatic index and plasma testosterone levels. Pituitary salmon gonadotropin-releasing hormone content and luteinizing hormone content were significantly lower in the melatonin-treated group on Day 60 and 90, respectively. These results indicate that the plasma melatonin profile is important for mediating photoperiodic signals that regulate brain-pituitary-gonadal axis in underyearling precocious male masu salmon.

We investigated the involvement of MCH in food intake in barfin flounder. The structure of barfin flounder MCH was determined by cDNA cloning and mass spectrometry. In fasted fish, the MCH gene expression and the number of MCH neurons in the brain were greater than controls. In white-reared fish, the MCH gene expression and the number of MCH neurons in the brain were greater than black-reared fish. Furthermore, white-reared fish grew faster than black-reared fish. These results indicate that a white background stimulated production of MCH and MCH, in turn, enhanced body growth, probably by stimulating food intake.