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In fish, individual photoreceptor cells in the pineal organ and retina contain complete melatonin rhythm generating systems. In the pike and seabream, this includes a photodetector, circadian clock, and melatonin synthesis machinery; the trout lacks a functional clock. The melatonin rhythm is due in part to a nocturnal increase in the activity of the arylalkylamine N-acetyltransferase (AANAT) which is inhibited by light. Two AANATs have been identified in fish: AANAT1, more closely related to AANATs found in higher vertebrates, is specifically expressed in the retina; AANAT2 is specifically expressed in the pineal organ. We show that there is a physiological day/night rhythm in pineal AANAT2 protein in the pike, and that light exposure at midnight decreases the abundance of AANAT2 protein and activity. In culture, this decrease is blocked by inhibitors of the proteasomal degradation pathway. If glands are maintained under light at night, treatment with these inhibitors increases AANAT2 activity and protein. Organ culture studies with the trout and seabream also indicate that the light-induced decrease of AANAT2 activity is prevented when proteasomal proteolysis is blocked. A cAMP-dependent pathway protects AANAT2 protein from degradation. These results provide a clue to understanding how light regulates the daily rhythm in melatonin secretion in fish photoreceptor cells and provides evidence that proteasomal proteolysis is a conserved element in the regulation of AANAT in vertebrates.

Serum T(3) (3,5,3' triiodothyronine) and serum T(4) (thyroxine) concentrations were repetitively assayed by radioimmunoassay over a three-year period in two male and two female immature captive whitetip reef sharks, Triaenodon obesus. These sharks were maintained at the Waikiki Aquarium, Honolulu, Hawaii, in an open system holding pool receiving 568 liters per minute of water from a saltwater well with an iodide concentration of 0.076 mg/liter. No significant male-female difference was observed for either serum T(3) or serum T(4). No seasonal pattern of serum T(3) was detected (P = 0.07). Serum T(3) concentrations ranged (mean +/- SEM) from 0. 52 to 0.83 ng/mL (0.67 +/- 0.01; n = 64). A significant seasonal difference was observed for serum T(4) (P < 0.001). Serum T(4) concentration was higher in winter (October-January) with a mean (range +/- SEM) of 6.58 ng/mL (1.48-8.77 +/- 0.35; n = 24) and lower in summer (May-August) with a mean of 3.62 ng/mL (1.34-5.71 +/- 0. 22; n = 24). The thyroid hormone T(4) has a seasonal rhythm even in immature sharks and may have an important role in physiology. J. Exp. Zool. 284:500-504, 1999.

Captive whitetip reef sharks, Triaenodon obesus,at Sea Life Park (SLP) Hawaii, Waimanalo, Hawaii develop goitre, whereas, T. obesus at the Waikiki Aquarium (WAQ), Honolulu,Hawaii do not develop goitre. To determine theeffects of natural concentrations of iodine on thereduction of goitre, two sharks from SLP with goitrewere placed in an enclosed coastal lagoon with naturalseawater. Using ultrasound measurements the twogoitres were initially 11.4 cm and 14.6 cm in depthand after 3 months decreased to 4.7 cm and 5.7 cm. Radioimmunoassay analysis of the thyroid hormoneT3 sera concentrations were initially 0.22 ng/mland 0.33 ng/ml and increased to 1.84 ng/ml after beingplaced in the lagoon. Sera T4 were initially0.93 ng/ml and 0.99 ng/ml and increased dramaticallyto 17 ng/ml and 56 ng/ml. Over the six month samplingperiod, two resident sharks in the lagoon with normalthyroids had sera T3 concentrations from 0.89ng/ml to 1.1 ng/ml, and sera T4 concentrationsfrom 3.1 ng/ml to 7.9 ng/ml. The hypothyroidcondition in the SLP goitred-sharks is likely linkedto the low environmental iodide (

In the present study, we examined the long-term effects of environmental salinity, diet (35% and 25% crude protein) and 17α-methyltestosterone (MT) on corresponding levels of pituitary and serum growth hormone (GH) and prolactins (tPRL177 and tPRL188) in the tilapia (Oreochromis mossambicus). We observed no discernible patterns in serum GH that would suggest an effect of salinity, diet or MT. However, serum GH levels in all treatments declined at 1 and 3h after first feeding. Serum tPRL177 and tPRL188 were significantly higher in freshwater (FW) than in seawater (SW) and levels were significantly affected by dietary protein. tPRL177 levels were higher in all groups fed a 35% protein diet, but tPRL188 levels were higher only in the groups fed the MT-treated 35% protein diet; only serum tPRL188 levels were affected by MT. Moreover, serum tPRL177 and tPRL188 increased throughout the sampling time-course. Subsequent work using fasted tilapia suggests that first feeding is likely to initiate the post-prandial suppression of serum GH levels. In contrast with the picture observed in blood, pituitary glands of SW animals showed higher levels of GH than FW fish. Pituitary GH was elevated by MT in both FW and SW. We also observed that pituitary tPRL177 and tPRL188 levels were higher in FW fish than in SW fish; tPRL177 and tPRL188 levels were elevated by MT only in FW animals. To assess the somatomedin activity of plasma from FW- and SW-reared tilapia, we measured [35S]-sulfate incorporation into ceratobranchial cartilage explants in vitro. Plasma from SW-adapted tilapia showed greater activity in this assay than plasma from FW-reared tilapia, suggesting that the GH-dependent IGF bioactivity of plasma is higher in SW-reared tilapia. Collectively, these studies suggest that the growth-promoting actions of SW rearing and of MT administration in tilapia may be linked to elevations in GH and/or prolactin (tPRL177 and tPRL188)levels.

The effects of long-term 17α-methyltestosterone (MT) treatment (10 mg MT kg −1 feed) and ration size on the growth of the euryhaline tilapia, Oreochromis mossambicus , in fresh water (FW) and seawater (SW) were examined. Tilapia were fed a limited daily ration, starting initially at 18% of body weight for yolk-sac fry and decreasing gradually to 4% at 126 days. Eight treatments were employed. Control and MT treatment groups were replicated in FW and SW using either fish fed the limited daily ration described above or fish fed twice that ration. On limited ration, control fish in SW grew to twice the size of their FW counterparts. Treatment with MT increased growth in both FW and in SW tilapia. Doubling the feeding ration produced a significant increase in growth in SW, but not in FW fish. The greatest growth was observed in SW, MT-treated fish on double ration which grew to >4.2 × the size of FW control fish on a limited ration. Routine metabolic rate was measured in tilapia reared from the yolk-sac fry stage in FW or SW. The rate of oxygen consumption in the SW tilapia was 72.4 ± 11.3 mg kg −1 h −1 (mean ± SE, n = 6) and 148.2 ± 9.2 mg kg −1 h −1 in FW fish ( n = 6). These data suggest that the reduction in routine metabolism in O. mossambicus in SW may account for the increase in growth of the SW animals over their FW counterparts.

In this study we examined the physiological responses of different strains of gilthead sea bream (Sparus aurata) to chronic and acute stress factors. Blood glucose levels, hematocrit, and lactate concentrations were examined in yearlings of different strains of gilthead sea bream subjected to different stress situations. In the first experiment, fish of the Ardag and ‘ebony’ strains were kept 45 days in moderate (35-45 kg/m3) or high (70-90 kg/m3) density. There were no significant effects on the blood glucose level but, at both densities, hematocrit was significantly higher in the Ardag strain (33.2±3.0% at moderate and 44.5±1.6% at high) than in the ebony strain (22.0±3.0% and 19.9±2.7%, respectively). The interaction of ‘strain’ and ‘crowding’ was highly significant, suggesting that hematocrit may respond differently in each strain to crowding. In the second experiment, we examined the effects of acute stress (handling without anesthesia) among four strains representing Mendelian mutations that affect body coloration in gilthead sea bream: ebony, ‘white’, ‘yellow’, and the normally pigmented Ardag. Significant variations in the glucose and lactate concentrations suggest that genotypes of gilthead sea bream may have heritable differences in their physiological responses to stress factors.

Pineal serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase; AANAT) is considered the key enzyme in the generation of circulating melatonin rhythms; the rate of melatonin production is determined by AANAT activity. In all the examined species, AANAT activity is regulated at the post-translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real-time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock-controlled regulation of seabream aanat2. DNA constructs were prepared in which green fluorescent protein was driven by the aanat2 promoters of seabream and Northern pike. In vivo transient expression analyses in zebrafish embryos indicated that these promoters contain the necessary elements to drive enhanced expression in the pineal gland. In the light-entrainable clock-containing PAC-2 zebrafish cell line, a stably transfected seabream aanat2 promoter-luciferase DNA construct exhibited a clock-controlled circadian rhythm of luciferase activity, characteristic for an E-box-driven expression. In NIH-3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL/CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E-box (i.e. the binding sites for BMAL/CLOCK and OTX5 that have been previously associated with pineal-specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms.

Serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase, AANAT) is the key enzyme in the generation of melatonin rhythms in the pineal gland and retinal photoreceptors. Rhythmic AANAT activity drives rhythmic melatonin production in these tissues. Two AANATs, AANAT1 and AANAT2, are present in teleost fish species. Different spatial expression patterns, enzyme kinetics and substrate preferences suggest that they may have different functions. Enzyme activity assays revealed that recombinant seabream and zebrafish AANAT1s, but not AANAT2s, acetylate dopamine with kinetic characteristics that are similar to those for tryptamine acetylation. High performance liquid chromatography analysis of seabream retinal extracts indicated the presence of N-acetyldopamine. Time-of-day analysis of retinal AANAT activity and concentration of melatonin, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and N-acetyldopamine revealed a daily pattern of retinal melatonin and N-acetyldopamine production that are correlated with retinal AANAT1 activity. In situ hybridization analysis of seabream retinal sections indicated that tyrosine hydroxylase is expressed in the inner nuclear layer (INL) and that AANAT1 is expressed in the outer nuclear layer (ONL) and INL. Together, these observations point to the possibility that dopamine is acetylated by retinal AANAT1 in the INL. Such novel activity of AANAT1 may reflect an important function in the circadian physiology of the retina.

17 pages, 6 figures, uropean sea bass Dicentrarchus labrax of the north-western (NW) and south-eastern (SE) Mediterranean Sea strains were exposed to different temperatures (13, 17 or 21° C) during the larval rearing (11–51 days post hatching, dph) or nursery periods (55–95 dph), in order to examine the effects of temperature on sex differentiation and subsequent growth during the first year of life. Higher growth was observed during exposure to higher temperatures, but fish of the NW strain exposed to 13 or 17° C during larval rearing exhibited compensatory growth once exposure to the lower temperatures finished, and as a result their final size at 300 dph was similar or greater to the group exposed to 21° C. Fish exposed to 17° C during the nursery period also had similar size to fish exposed to 21° C after 300 days of rearing, but the fish exposed to 13° C remained significantly smaller (ANOVA, n = 55–100, P < 0·05). There were significant differences in the sex ratio among the fish exposed to different temperatures during the two periods of rearing, with high temperature (21° C) resulting in a significantly higher percentage of males in the population, both in the NW (ANOVA, n = 2, P < 0·04) and SE populations (ANOVA, n = 2, P < 0·01). The masculinization effect of high temperature was significantly stronger during the larval rearing stage, both in the NW (ANOVA, n = 2, P < 0·005) and SE populations (ANOVA, n = 2, P < 0·01). None of the temperature manipulations could produce 100% females, suggesting that there is a part of the genetic component in sex differentiation which is not labile to environmental influence., This work was undertaken as part of "Improving production efficiency of sea bass farming by developing methodologies to eliminate environmental androgenesis", a research grant from the European Community to S.Z., C.C.M., F.P., and A. T. (contract Q5RS-2000-31365).

Serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase, AANAT) is the key enzyme in the biosynthesis of melatonin in the pineal gland and retinal photoreceptors. Rhythmic AANAT activity drives rhythmic melatonin production in these tissues. The presence of two AANATs, AANAT1 and AANAT2, has been previously demonstrated in three fresh water teleosts. This duality, the result of early gene duplication, is unique to teleost species. In this study, the cDNAs encoding for AANAT1 and AANAT2 were cloned from a marine fish, the gilthead seabream (sb, Sparus aurata). Northern blot hybridization analysis indicates that sbAANAT1 and sbAANAT2 are exclusively expressed in the retina and pineal gland, respectively. Bacterially expressed recombinant sbAANATs exhibit differential enzyme kinetics. Recombinant retinal sbAANAT1 has relatively high substrate affinity and low activity rate; it is inhibited by high substrate and product concentrations. In contrast, recombinant pineal sbAANAT2 exhibits low substrate affinity and high activity rate and is not inhibited by substrates or products. The two recombinant enzymes also exhibit differential substrate preference. Retinal sbAANAT1 acetylates a range of arylalkylamines while pineal sbAANAT2 preferentially acetylates indoleethylamines, especially serotonin. The different spatial expression patterns, enzyme kinetics, and substrate preferences of the two sbAANATs support the hypothesis that, as a consequence of gene duplication, teleosts have acquired two AANATs with different functions. Pineal AANAT2 specializes in the production of large amounts of melatonin that is released into the circulation and exerts an endocrine role. Retinal AANAT1, on the other hand, is involved in producing low levels of melatonin that execute a paracrine function. In addition, retinal AANAT1 may carry out an as yet unknown function that involves acetylation of arylalkylamines other than serotonin.

The melatonin-generating system of the European sea bass, Dicentrarchus labrax, was studied in vitro using a perfusion tissue culture methodology. The examined properties included photic entrainment in the pineal gland, and circadian rhythmicity in alternating dark and light, in contin- uous darkness, and in continuous light. Pineal glands were removed from adult fish and placed in a perfusion tissue culture at a constant temperature of 24°C. Melatonin discharge into the cul- ture medium was measured using radioimmunoassay. Melatonin concentrations during a pho- toperiod of 12h light:12h dark were low during the light periods and high during the dark. The melatonin discharge was inhibited by continuous light but strong and rhythmic in continuous dark with a circadian period (Tau) of about 24 h (n = 10). The results demonstrate that the European sea bass pineal gland is photosensitive, with ability to be entrained, and contains an internal cir- cadian oscillator that regulates melatonin production.

Effects of environmental salinity and 17α-methyltestosterone (MT) on growth and oxygen consumption were examined in the tilapia, Oreochromis mossambicus . Yolk-sac fry were collected from brood stock in fresh water (FW). After yolk-sac absorption, they were assigned randomly to one of four groups: FW, MT treatment in FW, seawater (SW) and MT treatment in SW. All treatment groups were fed to satiation three times daily. The fish reared in SW (both control and MT-treated groups) grew significantly larger than either group in FW from day 43 throughout the experiment (195 days). The fish fed with MT added to their feed grew significantly larger than their respective controls from day 85 in FW and in SW until the end of the experiment. The routine metabolic rate (RMR) was determined monthly from month 2 (day 62) to month 5 (day 155). A significant negative correlation was seen between RMR and body mass in all treatment groups. Among fish of the same age, the SW-reared tilapia had significantly lower RMRs than the FW-reared fish. The MT-treated fish in SW showed significantly lower RMRs than the SW control group at months 3–5, whereas MT treatment in FW significantly increased the RMR at month 3. Comparison of regression lines between RMR and body mass indicates that MT treatment in FW caused a significant increase in oxygen consumption at a given mass of the fish, whereas MT treatment was without effect on RMR in SW-reared fish. These results clearly indicate that SW-rearing and MT treatment accelerate growth of tilapia, and that RMR decreases as fish size increased. It is also likely that the increased RMR and growth in MT-treated tilapia in FW may be due to the metabolic actions of MT, although the reason for the absence of MT treatment in SW is unclear.

The effect of dietary arachidonic acid (ArA) on survival, growth, and cortisol level in different-age gilthead seabream larvae exposed to handling or daily fluctuating salinity was tested. Premetamorphosing (3–19 DPH) larvae were reared in 400-l V-tanks and fed one of three rotifer treatments containing ArA levels of 1.14, 2.11, or 3.87 mg g−1. At 20 DPH, the larvae were divided into two groups where each larval group was randomly divided over twelve 27-l aquaria (300 larvae per aquarium) and the stress of transfer was defined as an acute stressor. In each set of 12 aquaria, larvae were fed over 12 days three Artemia metanauplii treatments, which were tested in four aquaria per treatment, giving Artemia ArA levels of 0.59, 3.42, or 5.86 mg g−1 dry weight (DW). One set of 12 aquaria received seawater of constant salinity (25‰) and these larvae, exposed only to the stress of transfer, were considered as controls. The other set of 12 aquaria was supplied with seawater with a daily fluctuating salinity from 25‰ to 40‰ and back to 25‰, exposing the larvae to 12 days of salinity change. Another trial was carried out on 30 DPH metamorphosing larvae, which were similarly stocked in the aquaria and tested with the identical Artemia treatments as the premetamorphosing larvae study. A positive correlation was found between increasing dietary ArA level and survival at the end of the study in the control premetamorphosing (20–32 DPH) and metamorphosing (30–42 DPH) larvae (38%, 48.6%, and 77.2%, and 58%, 56.4%, and 90.4%, respectively). On the other hand, premetamorphosing and metamorphosing larvae exposed to salinity change, although exhibiting an increase in survival at the intermediate level of ArA, demonstrated a decrease in survival (55.3%, 60.0%, and 25.8%, and 70%, 83%, and 76%, respectively) when fed the highest ArA level. In the control metamorphosing larvae, basal cortisol (6.0, 8.2, and 11.4 ng g−1 DW) was independent of dietary ArA while fish exposed to salinity change demonstrated distinctly higher basal cortisol levels (7.5, 15.9, and 19.8 ng g−1 DW) that markedly increased with rising dietary ArA levels at 42 DPH. Fish exposed to salinity change and fed Artemia containing 0.59, 3.42, and 5.86 mg g−1 ArA exhibited significantly (P

The fish pineal organ, through its 24 h rhythmic release of melatonin, acts as a transducer of the photoperiod, influencing different physiological functions (e.g. reproduction, growth). We have investigated the binding of 2-[125I]iodomelatonin to whole brain membrane preparations from pikes (Esox lucius L., teleost) maintained for 24-48 h under different photoperiodic conditions. Specific binding was stable, reversible, saturable and sensitive to the presence of a GTP analogue. Scatchard analysis revealed one class of binding sites. Displacement experiments suggested the presence of two components with affinities in the femtomolar and nanomolar range of concentrations, respectively. The Bmax exhibited monophasic nycthemeral variations, with higher values at the light-to-dark transition (34.0 +/- 4.5 fmol/mg protein) and low values during the second half of night (10.0 +/- 1.0 fmol/mg protein). Under the same conditions, the KD exhibited biphasic variations: values were low during daytime and at the middle of the dark phase (approximately 100 pM); they were high at the beginning (approximately 225 pM) and at the end (approximately 330 pM) of the night. These variations were maintained under constant light (LL) and constant darkness (DD). Thus, the variations in the number and affinity of the melatonin binding sites were controlled by circadian oscillators, synchronized by the photoperiod. The nature of these oscillators is not known. Therefore, in fish, we suggest that the photodependent effects of melatonin result from the circadian variations of both its production by the pineal and its binding sites in the brain.

Distribution and metabolism of the thyroid hormone 3,5, 3'-l-triiodothyronine (T3) were studied in several ways to gain insights into these processes in the warm water fish tilapia Oreochromis mossambicus. Trace doses of 125I-labeled T3 (T*3)1 were injected intraarterially, extraarterially, or intraperitoneally in freshwater-reared male tilapia to explore plasma clearance kinetic responses to these different input modalities. Multicompartmental analysis of the plasma clearance data indicated a kinetic distribution of T*3 much like that reported for the rat and human, with about 2% of total body T*3 in plasma, 5% in rapidly exchanging tissues such as kidney and liver, and 93% in slowly exchanging tissues such as muscle. However, plasma clearance rates (PCR, 5.37 mL/h . 100 g body wt) and plasma appearance rates (PAR3 = PCR x [T3] plasma = 36.3 ng/h . 100 g body wt) were quite different than these indices in rat and human and 5 to 50 times larger than values reported for rainbow trout. On a whole-body basis, normalized for body weight, the tilapia we studied produced and accumulated much more T3 than rat, human, or rainbow trout. Enzymatic and chromatographic analyses of the plasma clearance data samples indicated substantial production of labeled glucuronide, but not sulfate, conjugates of iodothyronines (TiG) of unknown origin appearing in plasma. The TiG appeared beginning a few hours postinjection, peaked at 6 hours, and yielded a predicted steady-state TiG level of 8.3% of the T3 level in plasma. In contrast, in published studies, no conjugates were detected in rainbow trout plasma from 2 to 24 h after iv injection of T*3, T*4, or reverse-T*3, although conjugates of all were present in bile. To our knowledge, although T3 and T4 sulfate conjugates are present in the sera of several mammals, this is the first quantification of iodothyronine glucuronides reported in blood of any species under normal conditions. This might have physiological significance for the tilapia, with T3G providing a reversible storage form of T3 in blood, as has been suggested for sulfate conjugates of T3 and T4 in blood of several mammals.

Plasma volumes in male tilapia (Oreochromis mossambicus) of different size were estimated following intracardial injection of radioiodinated human serum albumin ((125)I-HSA), coupled with short-term, early sampling transient response analysis of 1251-HSA disappearance from the plasma pool. This approach circumvents vascular marker leakage problems associated with constant steady state indicator dilution methods, minimizes some sampling and mixing problems, and simplifies analysis of the data. Changes in hematological parameters due to experimental stress were also studied, because the fish were not chronically cannulated. Results were used in a novel way to correct estimates of plasma volume upward by 15%, thereby providing a potentially useful alternative approach to vascular volume measurement in species where stress-eliminating or reducing techniques, e.g., cannulation, are impractical or infeasible. Hematrocrits increased 38% at the onset, from 24.9% to 34.4%, and remained essentially constant during the 60 minute kinetic study, and plasma osmolalities increased 7%. Corrected plasma volumes Vp (ml) were a linear function of body weight (BW). The group mean Vp was 2.93% of BW and corresponding blood volumes were 3.9% of BW.