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The Effect of Photoperiod on Diel Rhythms in Serum Melatonin, Cortisol, Glucose, and Electrolytes in the Common Dentex,Dentex dentex
General and Comparative Endocrinology 113, 240–250 (1999) Article ID gcen.1998.7190, available online at http://www.idealibrary.com on
The Effect of Photoperiod on Diel Rhythms in Serum Melatonin, Cortisol, Glucose, and Electrolytes in the Common Dentex, Dentex dentex M. Pavlidis,*,1 L. Greenwood,† M. Paalavuo,‡ H. Mo¨lsa¨,‡ and J. T. Laitinen§ *Department of Aquaculture, Institute of Marine Biology of Crete, P.O. Box 2214, GR-710 03, Heraklio, Crete, Greece; †Lowestoft Laboratory, Centre for Environment, Fisheries, and Aquaculture Science, Lowestoft, Suffolk NR33 OHT, England; ‡Department of Applied Zoology and Veterinary Medicine, University of Kuopio, P.O. Box 1627, FIN-702 11, Kuopio, Finland; and §Department of Physiology, University of Kuopio, P.O. Box 1627, FIN-702 11, Kuopio, Finland Accepted September 25, 1998
first part of the scotophase under all regimes, except the 16L:8D where no diel rhythmicity was detected. During the photophase, cortisol was positively correlated with glucose, Naⴙ, and Clⴚ and negatively with Kⴙ. During the scotophase, melatonin was positively correlated with glucose and electrolytes. Results indicated that cortisol may be responsible for the observed rhythmicity of glucose and that melatonin may play a role in glucose and ion regulation in common dentex. r 1999 Academic Press Key Words: diel rhythms; cortisol; common dentex (Dentex dentex); electrolytes; glucose; melatonin; photoperiod.
Diel rhythms in serum concentrations of melatonin, cortisol, glucose, sodium, chloride, and potassium were studied in the common dentex, Dentex dentex, under different photoperiods (DD, 8L:16D, 12L:12D, 16L:8D). Photoperiod affected both the diel rhythms and the absolute values of the estimated blood components. Regardless of the photoperiod, melatonin titers were elevated during the scotophase (384.3 ⴞ 13.9 pg/ml) compared with a mean baseline level of 54.4 ⴞ 2.7 pg/ml during the photophase. Serum melatonin concentrations reflected the prevailing photoperiod and constantly elevated melatonin levels with no diel rhythmicity were evident in fish held in the DD protocol. A circadian-like pattern in serum cortisol was observed in fish that were kept at the DD and 8L:16D protocols with cortisol peak at 18:00 h in the night. Fish exposed to the 16L:8D regime showed highest cortisol levels at 10:00 h, while no rhythmicity was evident under the 12L:12D protocol. A phase shift of 4 h between the peaks of cortisol and glucose was evident in fish exposed to the DD, 8L:16D, and 12L:12D regimes. Diel patterns of changes in serum Naⴙ and Clⴚ were observed only in the fish held in the DD protocol. Serum Kⴙ values were lowest during the
All vertebrates display a similar diel pattern of changes in plasma melatonin levels in response to conventional light-dark cycles, with baseline levels during the photophase and high concentrations during the scotophase (Rollag and Niswender, 1976; Pang et al., 1977; Gern et al., 1978; Underwood, 1985; Armstrong and Redman, 1993; Stokkan et al., 1995). In addition, the role of melatonin in conveying information on the diurnal and seasonal changes in daylength has been reported in various fish species: the rainbow trout, Oncorhynchus mykiss (Gern et al., 1978; Randall et al., 1991; Alvarin˜o et al., 1993); brook trout, Salvelinus fontinalis (Zachmann et al., 1992a); Atlantic salmon, Salmo salar (Randall et al., 1995); common carp, Cypri-
1 To whom correspondence should be addressed at Department of Aquaculture, Institute of Marine Biology of Crete, P.O. Box 2214, GR-710 03, Heraklion, Crete, Greece. Fax: 081-241882. E-mail: [email protected].
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nus carpio (Kezuka et al., 1988); pike, Esox lucius (Falco´n et al., 1989); goldfish, Carassius auratus (Kezuka et al., 1992); white sucker, Catostomus commersoni (Zachmann et al., 1992b); and catfish, Silurus asotus (Iigo et al., 1997). Although the role of melatonin in transducing the information about ambient photoperiodic changes into rhythmic bodily function such as circadian and seasonal rhythms and in mediating several light-cued physiological processes such as locomotory activity (Kavaliers, 1980) and pigmentation (Gern et al., 1980) is well documented, the action of melatonin in regulating metabolic and/or endocrine function in fish is unclear. Melatonin treatment significantly reduces the hypothalamic noradrenaline content in Chana punctatus (Khan and Joy, 1988), is hypoglycemic in sham-operated but not pinealectomized goldfish, and increases liver glycogen levels in sham-operated but not pinealectomized goldfish, C. auratus (Delahunty et al., 1978). Interactions among cortisol, arginine vasotocin (hormones closely associated with electrolyte balance), and melatonin have been reported in teleosts (Delahunty et al., 1977; Kulczykowska, 1995). In addition, a similar pattern of changes in plasma titers of melatonin, sodium, and chloride during seawater adaptation of coho salmon has been reported by Folmar and Dickoff (1981), indicating a possible role of melatonin in osmoregulation and/or stress adaptation in seawater. Data on stenohaline marine species are lacking. The common dentex, Dentex dentex (L., 1758) is a common marine fish whose melatonin secretory profiles and diel rhythms of serum components are unknown. The objectives of the present study were to (1) describe the daily melatonin rhythms in the blood of the D. dentex maintained under different photoperiodic regimes; and (2) examine possible relationships between melatonin rhythms and other serum components (cortisol, glucose, sodium, potassium, chloride) implicated in osmoregulation in this stenohaline marine species.
MATERIALS AND METHODS Animals One hundred twenty immature 9-month-old common dentex weighing 140.2 ⫾ 8.2 g, reared under
ambient photothermal conditions at the Aquaculture Research Station of the IMBC, were transferred to indoor laboratory facilities, in February 1996. Fish were distributed randomly into two 6 ⫻ 500 L circular tanks (10 fish/tank) supplied with continuously aerated and running seawater (water flow, 8–10 L/min; dissolved O2, 4.5–6.0 mg/L, salinity, 38–40%). Fish were fed once a day by hand with wet pellets (50% raw fish, 50% commercial pellets). On the day of sampling no food was given. Black plastic curtains surrounded each set of tanks and artificial illumination was achieved using fluorescent strip lights located 130 cm above the water surface, controlled by electric time clocks with no twilight period. Light intensity was 150 lx at the water surface (tank depth: 1 m).
Experimental Design Fish were acclimated to each respective photoperiod treatment for 5–6 weeks prior to sampling. In all experiments dawn was set at 06:00 h. Sampling began at 06:00 h and was repeated at 4-h intervals (06:00, 10:00, 14:00, 18:00, 22:00, 02:00 h) over a 24-h cycle. Ten fish were used at each time interval. To minimize stress, 10 min prior to sampling the level of the water was reduced (by siphoning) and anesthetic (0.25 ml/L Ethylene glycol monophenyl ether, Merck, Hohenbrunn) was added to the tank water. Lightly anesthetized fish were netted, their head was covered with a towel, and blood was withdrawn from the caudal vessel. Clotted blood was centrifuged at 2500g and serum was collected, aliquoted for the different assays, and stored at ⫺20°C until analysis. Sampling took 15–20 min per tank. When blood collection was completed fish were placed in 70-L tanks provided with oxygen for recovery and then were returned to the experimental tanks. Two series of experiment were performed: A. In the first, fish (n ⫽ 60) were exposed to 12L:12D light-dark cycle (water temperature 15.6 ⫾ 0.3°C, sampling on April 2) and then subjected to 16L:8D regime (17.0 ⫾ 0.8°C, sampling on May 10). B. In the second, fish (n ⫽ 60) were subjected to 8L:16D light-dark regime (sampling on April 2) and then placed on constant darkness (DD) (sampling on May 10).
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say of diethyl ether-extracted serum samples as described by Scott et al., (1984). Briefly, serum aliquots were shaken with 4 ml of diethyl ether. The serum was frozen in liquid nitrogen and the ether decanted into a 12 ⫻ 75-mm borosilicate glass tube. The ether was then evaporated and the residue containing free cortisol redissolved in assay buffer. Aliquots of 50 or 100 µl of these diluted samples were used in the radioimmunoassay. The cortisol antibody was purchased from Scottish Antibody Production Unit (Strathclyde, Scotland) and tritiated cortisol purchased from Amersham International Ltd. (UK). The inter- and intraassay coefficients of variation were 5.1% (n ⫽ 14) and 1.4% (n ⫽ 20), respectively. Glucose was estimated by the enzymatic colorimetric (GOD/PAP) method (Biosis, Greece). Serum electrolytes were determined by the use of ion-selective electrodes (Ciba Corning 644 Na⫹/K⫹/Cl⫺ analyzer).
Analytical Techniques Melatonin was analyzed from chloroform-extracted serum samples using a specific RIA (Fraser et al., 1983; Valtonen et al., 1993). Briefly, duplicate samples of serum, melatonin standards (5–200 pg/ml), and lowand high-melatonin control samples (11 and 66 pg/ml, respectively) in a total volume of 0.5 ml were extracted into 3.0 ml of chloroform. The dried residue was reconstituted in phosphate-buffered saline, pH 7.0, containing 1 g of gelatin per liter and assayed for melatonin using the G/S/704-8483 antiserum (Stockgrand Ltd, University of Surrey, Guildford, Surrey UK) and tritiated melatonin as a tracer. Displacement curves (three different pools of dentex serum, one daytime pool and two nighttime pools, with three different serum volumes, day pools 25, 50, and 100 µl; night pools 10, 25, and 50 µl) demonstrated linear parallelism after logit-log transformation (standard curve, r2 ⫽ 1.000, slope ⫽ ⫺0.995; day pool, r2 ⫽ 1.000, slope ⫽ ⫺0.952; night pool 1, r2 ⫽ 0.99, slope ⫽ ⫺0.943; night pool 2, r2 ⫽ 0.999, slope ⫽ ⫺0.942). The recovery of melatonin (10, 20, or 50 pg) added to dentex serum was 96.1 ⫾ 3.0% (mean ⫾ SEM, n ⫽ 3). The intraassay coefficients of variation were 6.5 and 3.1% for melatonin concentrations of 11 and 66 pg/ml, respectively (n ⫽ 6). The interassay coefficients of variation for these melatonin concentrations were 11.6 and 5.2%, respectively. Cortisol levels were determined by radioimmunoas-
Statistical Analysis Data are expressed as means ⫾ standard error. However, for the melatonin data of Table 1, the mean, standard deviation, range, and intragroup coefficient of variation (CV%) are presented to illustrate the fact that not a single individual fish in the DD group exhibited low daytime serum melatonin concentrations at any time point studied. The KolomogorovSmirnov test was applied to check for a normally
TABLE 1 Dentex Serum Melatonin Concentrations (pg/ml), Mean, SD, Range, and CV at Each Sampling Point and Photoperiod Regime 12L:12D
Hour of sampling
Mean
SD
06:00 10:00 14:00 18:00 22:00 02:00
58.0 68.7 35.3 267.2 282.3 224.5
21.9 31.3 13.7 91.7 83.7 52.2
06:00 10:00 14:00 18:00 22:00 02:00
50.9 52.6 226.7 330.5 327.5 247.8
19.0 28.1 129.8 123.0 65.5 64.0
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Range 39–106 40–154 21–54 155–480 145–385 132–280 8L:16D 29–87 19–101 81–403 188–596 269–459 154–338
16L:8D CV (%)
Mean
SD
Range
CV (%)
38 45 39 34 30 23
64.8 66.9 46.5 44.0 329.6 484.1
30.6 29.2 12.3 6.9 138.6 101.1
20–95 30–134 29–69 31–53 106–570 311–685
47 44 26 16 42 21
37 53 57 37 20 26
458.7 508.0 452.7 427.4 604.9 579.1
90.6 150.0 98.7 95.6 176.9 168.5
329–630 234–797 354–641 274–627 337–826 304–869
20 29 22 22 29 29
DD
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distributed population and the Bartlett test was applied to verify the homogeneity of variances. One-way analysis of variance (ANOVA) or the Kruskal-Wallis test (when variances were not homogeneous) was used to test for significant changes between sampling times within a single photoperiod or between the different photoperiod regimes within a single serum component. If significant (P ⬍ 0.05), Tukey’s significant means test or Dunn’s multiply comparison test was applied to compare mean values at different sampling times. Comparison of the estimated serum parameters between the photo- and scotophase within each respective photoperiod protocol was performed by the t test or the Mann-Whitney rank sum test. Pearson product moment correlation was applied to measure the strength of association between pairs of the estimated variables.
fish held in the other experimental photoperiods (Table 1). Moreover, the intragroup coefficient of variation was similar between each group, indicating that melatonin concentrations in the DD group did not deviate from the mean more than individual values in other groups and time-points; this is in contrast to the situation where individual fish would exhibit nonsynchronous free-running melatonin rhythm with low daytime levels randomly found at each time point. Fish under the 8L:16D, 12L:12D, and 16L:8D photoperiods displayed a similar diel pattern of melatonin profile; minimum values occurred 4 h prior to lights off and maximum values within 0–4 h after lights off (Fig. 2). As the duration of photophase increased, the duration of elevated melatonin levels decreased, being shortest in fish held under the 16L:8D protocol. There was no significant effect of the order of sampling within a tank.
RESULTS
Cortisol
Melatonin Light significantly affected both the absolute values and the diel rhythms of serum melatonin concentrations. Regardless of the photoperiod regimen, melatonin levels were significantly higher during the scotophase (384.3 ⫾ 13.9 pg/ml) and returned to a mean baseline level of 54.4 ⫾ 2.7 pg/ml during the photophase (Fig. 1). No diel rhythm in serum melatonin was evident in fish held in the DD protocol. In particular, individual melatonin concentration at each sampling was significantly higher than the respective values in
FIG. 1. Serum melatonin (means ⫾ SEM) in the common dentex, Dentex dentex, during the photo- and scotophase of the experimental photoperiod protocols.
Significant fluctuations in serum cortisol were observed in fish under the 16L:8D, 8L:16D, and DD protocols, but not in the 12L:12D protocol (Fig. 3). Fish exposed to the long photoperiod (16L:8D) had the lowest cortisol levels in the middle of the dark period (02:00 h) and the highest levels 4 h after the lights on. In constant darkness (DD), serum cortisol levels were at a minimum between 02:00 and 06:00 h and at a maximum at 18:00 h. The fish on short photoperiod (8L:16D) displayed minimum cortisol values in the middle of the photophase (10:00 h) and a maximum between 18:00 and 22:00 h, 4 to 8 h after lights off. In the 12L:12D photoperiod, serum cortisol levels were relatively high at all time points examined with the exception of low levels at 06:00 h, but no significant rhythmicity was apparent. Regardless of photoperiod, there was a time lag of 8 to 12 h between the minimum and the maximum in cortisol level. There was no statistically significant difference in the absolute cortisol levels in fish held under the different photoperiod protocols (Table 2). However, in fish exposed to the 8L:16D and 16L:8D regimes, differences in the mean cortisol concentrations between the photo- and scotophase were observed; in the 8L:16D regime lower cortisol levels were found during the photophase (11.3 ⫾ 2.0 ng/ml) and higher during the scotophase (21.6 ⫾ 3.1 ng/ml), while in the 16L:8D there was an
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opposite pattern (photophase, 28.2 ⫾ 3.9 ng/ml; scotophase, 14.7 ⫾ 3.6 ng/ml). No significant effect of the order of sampling within a tank was observed.
Glucose There were diel rhythms in serum glucose levels in fish held in the DD, 8L:16D, and 12L:12D light-dark
FIG. 3. Photoperiod effect on serum cortisol levels in the common dentex, Dentex dentex, sampled at 4-h periods throughout the day. For details see legend of Fig. 2.
FIG. 2. Photoperiod effect on serum melatonin levels in the common dentex, Dentex dentex, sampled at 4-h periods throughout the day. Data are means ⫾ SEM (n ⫽ 10/sampling point). Dark horizontal lines indicate period of darkness (✬, lights-on; 䊉, lightsoff). Means with different letters differ significantly from one another, P ⬍ 0.05).
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cycles (Fig. 4). The lowest glucose levels occurred at 10:00 h for the DD and 12L:12D protocols or at 14:00 h for the 8L:16D protocol, and serum glucose concentrations attained maximum values during the scotophase (22:00 h). Higher absolute glucose concentrations were found in fish exposed to constant darkness (DD) than in fish held in the 8L:16D protocol (Table 2). No differences in the mean glucose levels between the photo- and scotophase were detected. An effect of the order of sampling with an increase in plasma glucose values with increasing order of sample from each tank
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Diel Rhythms of Serum Components in Dentex
TABLE 2 Absolute Values of Serum Parameters (Mean ⫾ SEM) in Dentex dentex Acclimated to Different Photoperiods Group
T (°C)
Cortisol (ng/ml)
Glucose (mmol/L)
Na⫹ (mmol/L)
K⫹ (mmol/L)
Cl⫺ (mmol/L)
DD 8L:16D 12L:12D 16L:8D
17.0 ⫾ 0.8 15.6 ⫾ 0.3 15.6 ⫾ 0.3 17.0 ⫾ 0.8
18.3 ⫾ 3.2 18.8 ⫾ 2.2 19.6 ⫾ 1.9 22.6 ⫾ 2.9
4.55 ⫾ 0.27a 3.54 ⫾ 0.16b 3.65 ⫾ 0.19 3.92 ⫾ 0.18
203.0 ⫾ 1.0a 196.8 ⫾ 0.6bc 199.1 ⫾ 0.7b 200.6 ⫾ 0.8a
2.94 ⫾ 0.07a 2.62 ⫾ 0.06c 2.51 ⫾ 0.06c 2.20 ⫾ 0.06b
186.2 ⫾ 0.8a 177.7 ⫾ 0.5c 178.4 ⫾ 0.7c 182.2 ⫾ 0.8b
Note. Means with different letters differ significantly from one another a–b, c–d (P ⬍ 0.05).
was observed in all protocols except for the 16L:8D regime.
Electrolytes Statistically significant changes in mean serum Na⫹ and Cl⫺ levels were seen only in fish exposed to constant darkness, in which maximum ion concentrations were observed at 22:00 h (Fig. 5). Under the other photoperiods no diel rhythmicity in Na⫹ and Cl⫺ was detected. There were higher absolute values for Na⫹ and Cl⫺ in fish exposed to the DD and 16L:8D photoperiods compared with those held under 8L:16D and 12L:12D protocols (Table 2). Significant diel patterns of serum K⫹ levels were found under all photoperiodic conditions, with the exception of the 16L:8D protocol. Fish held in the 8L:16D and 16L:8D protocols showed minimum potassium values at 18:00 h, while fish exposed to the DD regime exhibited a minimum at 06:00 h (Fig. 6). Higher absolute potassium values were found in fish held in the DD protocol whereas there were lower values in fish exposed to the 16L:8D photoperiod (Table 2). There were no differences in the mean values of the estimated serum electrolytes between the light and dark period of each treatment.
Correlation between the Serum Parameters In fish under the 8L:16D protocol, melatonin was positively correlated with cortisol (r ⫽ 0.523,P ⬍ 0.001), while in the 16L:8D regime a negative correlation was found (r ⫽ ⫺0.281, P ⬍ 0.05). When the photophase of all regimes was analyzed the following relationships were found: cortisol was positively correlated with glucose (r ⫽ 0.423, P ⬍ 0.001), Na⫹ (r ⫽ 0.341, P ⬍ 0.01), and Cl⫺ (r ⫽ 0.364, P ⬍ 0.001) and negatively with K⫹ (r ⫽ ⫺0.248, P ⬍ 0.05); glucose with Na⫹ (r ⫽ 0.308,
P ⬍ 0.01) and Cl⫺ (r ⫽ 0.313, P ⬍ 0.01). When the scotophase was analyzed significant positive correlations were observed between melatonin and glucose (r ⫽ 0.307, P ⬍ 0.001), Na⫹ (r ⫽ 0.353, P ⬍ 0.001), K⫹ (r ⫽ 0.221, P ⬍ 0.01), and Cl⫺ (r ⫽ 0.475, P ⬍ 0.001); glucose and Na⫹ (r ⫽ 0.411, P ⬍ 0.001), K⫹ (r ⫽ 0.348, P ⬍ 0.001), and Cl⫺ (r ⫽ 0.378, P ⬍ 0.001).
DISCUSSION The present study was conducted to gain basic information on the effects of different photoperiods on certain serum characteristics of the common dentex. This is the first study to document diel changes in serum melatonin, cortisol, glucose, and electrolytes concentrations in this fish. The experimental design aimed to overcome possible effects of stocking density, feeding regime, size, and sexual condition on the diel pattern of the estimated parameters. The validation experiments indicated that melatonin levels were accurately and quantitatively estimated from chloroform-extracted serum samples. In most mammals, daytime serum melatonin levels are typically ⬍10 pg/ml and during the hours of darkness, 50 and 150 pg/ml (Arendt, 1985). In the fish studied so far, circulating melatonin levels are clearly higher between 50 and 100 pg/ml during the photophase for the pike (Falco´n et al., 1989), the rainbow trout (Gern et al., 1978; Kukkonen and Laitinen, 1987), the Atlantic salmon (Bromage et al., 1995), and the common carp (Kezuka et al., 1988). In these studies, nightime melatonin levels typically fluctuated around 300–500 pg/ml with somewhat lower concentrations in some of the studies with rainbow trout. Thus the melatonin levels currently reported for the dentex closely resemble
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Pavlidis et al.
those found in other species of fish. A recent study revealed, however, that in the catfish on a 12L:12D photoperiod, maximal nocturnal plasma melatonin levels were only about 40 pg/ml (Iigo et al., 1997) suggesting that in fish, there may be marked speciesspecific differences in the diurnal rhythm of circulating melatonin concentration. Although photoperiod appears to be the major regulator of melatonin secretion in many warmblooded animals (Reiter, 1991), a significant effect of environmental temperature on the amplitude of mela-
FIG. 5. Photoperiod effect on serum sodium and chloride levels in the common dentex, Dentex dentex, sampled at 4-h periods throughout the day. For details see legend of Fig. 2.
FIG. 4. Photoperiod effect on serum glucose levels in the common dentex, Dentex dentex, sampled at 4-h periods throughout the day. For details see legend of Fig. 2.
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tonin rhythm has been clearly documented in poikilotherms (Underwood, 1985; Iigo and Aida, 1995; Randall et al., 1995). In the present study, peak melatonin levels were around 300 pg/ml in the two photoperiodic groups sampled in April (8L:16D and 12L:12D; water temperature, 15.6 ⫾ 0.3°C) and around 500 pg/ml when the same individuals were sampled again for the two other photoperiods in May (DD and 16L:8D; water temperature, 17.0 ⫾ 0.8°C). Although the temperature change between these two sampling sessions was relatively modest, these observations could reflect a temperature-dependent increase in the amplitude of melatonin secretion rhythm in the dentex. The present data demonstrate that plasma melatonin concentrations provide an accurate representation of the prevailing photoperiod in the dentex, suggesting that photoperiodic manipulations will result in
Diel Rhythms of Serum Components in Dentex
FIG. 6. Photoperiod effect on serum potassium levels in the common dentex, Dentex dentex, sampled at 4-h periods throughout the day. For details see legend of Fig. 2.
highly predictable changes in the duration of nocturnal melatonin signal in this fish species; this is similar to observations on other fish species (Iigo and Aida, 1995; Randall et al., 1995). Moreover, constantly elevated melatonin levels were evident in dentex held in DD, suggesting that in contrast to most teleosts, in which melatonin production is driven by a pineal clock, melatonin production in the dentex occurs primarly in response to darkness, as has been described in the trout (Gern and Greenhouse, 1988;
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Randall et al., 1991; Max and Menaker, 1992; Thibault et al., 1993; Bolliet et al., 1996; Begay et al., 1998). Diel rhythmicity of circulating cortisol has been extensively studied in freshwater fish species (Boujard and Leatherland, 1992a). However, the exact pattern of cortisol rhythm varies, with peak values occurring during the daylight hours (Garcia and Meier, 1973; Spieler and Noeske, 1984; Boujard and Leatherland, 1992b), during nighttime (Redgate, 1974; Rance et al., 1982; Pickering and Pottinger, 1983), or during both periods (Peter et al., 1978; Ku¨hn et al., 1986). A circadianlike pattern in circulating cortisol was observed in common dentex that were exposed to a short photoperiod or constant darkness with a cortisol acrophase at 18:00 h. A phase shift of the cortisol peak was evident in fish exposed to long photoperiods, with an acrophase at 10:00 h in the morning, in a phase with a period of hyperphagia. Shifts in acrophases of cortisol rhythms have been described for several other animals (Meier and Fivizzani, 1975; Pickering and Pottinger, 1983; Ku¨hn et al., 1986), possibly reflecting differences in light/dark cycle, temperature, or feeding activity. No diel rhythm in cortisol was detected in fish held in the 12L:12D regime. This could be partly explained by the high individual variability in serum cortisol levels or by the fact that the 4-h interval between samplings could have masked a brief peak. Similar high variability among ‘‘unstressed’’ individuals or episodic elevation of circulating cortisol has been reported in fish (Pickering and Pottinger, 1983; Nichols and Weisbart, 1984; Audet et al., 1986). Serum glucose levels peaked during the scotophase (22:00 h) in all regimes apart from the 16L:8D in which there was no diel glucose rhythm. Similar results, with glucose acrophase during the scotophase, have been reported in sea bream and sea bass (Pavlidis et al., 1997), red porgy (M. Pavlidis, M. Paspatis, M. Koistinen, T. Paavola, P. Divanach, and M. Kentouri, unpublished data), rainbow trout (Boujard and Leatherland, 1992a; Boujard et al., 1993), and common carp (Ku¨hn et al., 1986). A phase shift of 4 h in acrophase of cortisol and glucose was evident in fish exposed to the DD, 8L:16D, and 12L:12D regimes. Therefore, these results indicate that the serum glucose zenith in the common dentex does not reflect food absorption from the gut, but may rather reflect gluconeogenic activity due to elevated cortisol concentrations. An effect of sampling sequence
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with increased serum glucose levels with passage of time was evident in all experiments. Laidley and Leatherland (1988) reported similar results in a study in rainbow trout. This difference does not nullify the observed glucose fluctuations. It should, however, be taken into consideration when planning for future experiments monitoring serum glucose in this particular species. Diel changes in serum sodium and chloride were observed only in fish on the DD protocol. Lowest potassium values were found during the first part of the scotophase in all regimes, except the 16L:8D in which there was no diel rhythmicity. Seasonal or diel variations in monovalent ions, although not necessarily nor consistently circadian in periodicity, have been reported for sea bream (Pavlidis et al., 1997), sea bass (Carillo et al., 1986; Pavlidis et al., 1997), rainbow trout (Laidley and Leatherland, 1988), and common carp (Ku¨hn et al., 1986). These changes may reflect circadian variations in ion exchanges across the gills and/or urinary electrolyte excretion. However, the biological significance, if any, of the observed diel K⫹ fluctuations remains to be elucidated. Interestingly, higher absolute values of Na⫹ and Cl⫺ paralleled the high melatonin titers in the DD and 16L:8D regimes, possibly reflecting temperature-dependent increases in the amplitude of electrolyte rhythm in the dentex. Remarkable, photoperiod-related, relationships between the estimated parameters were noted in the common dentex. Glucose was positively correlated with electrolytes, indicating a close relation between glucose and ion regulation. More importantly, cortisol was positively correlated during the photophase with glucose, Na⫹, and Cl⫺ and negatively with K⫹, supporting the proposed role of cortisol in intermediary metabolism and osmoregulation in fish (Henderson and Garland, 1980). The absence of such a correlation at the scotophase may explain the lack of correlation in plasma cortisol and glucose values reported in other studies (Chan and Woo, 1978; Ku¨hn, et al., 1986; Laidley and Leatherland, 1988). Melatonin was positively correlated with glucose and electrolytes during the scotophase in all groups. In addition, melatonin was positively correlated with cortisol in the 8L:16D protocol, and negatively in the 16L:8D regime, indicating a possible role of melatonin in electrolyte balance and metabolic processes as a result of a differential
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Pavlidis et al.
response to light. Cortisol and prolactin are known to either influence melatonin production or be affected by melatonin in fish (Delahunty et al., 1977; deVlaming et al., 1974a,b; Gern et al., 1984). Folmar and Dickhoff (1981) also reported a relationship between the highest serum concentrations of melatonin and Na⫹ ⫺ Cl⫺ during seawater adaptation of coho salmon. Whether this correlation indicates a direct or indirect (through other endocrine regulatory systems, e.g., argininvasotocin) involvement of melatonin in ion regulation is still unknown.
ACKNOWLEDGMENTS The study has been carried out with financial support from the Commission of the European Communities, Agriculture and Fisheries (FAIR) specific RTD programme, CT-95-0407, ‘‘Common dentex, a prime new species for aquaculture. Development of methods for reliable egg production.’’ It does not necessarily reflect its views and in no way anticipates the Commission’s future policy in this area. We are indebted to Prof. M. Kentouri for helpful comments on the manuscript. The authors express their gratitude to Mrs. M. Symsiridou for valuable efforts in fish husbandry and technical help, to Mr. S. Kukkonen for technical assistance in melatonin determination, and to the staff of Aquaculture Department of IMBC for their collaboration in carrying out this study.
REFERENCES Alvarin˜o, J. M. R., Randall, C. F., and Bromage, N. R. (1993). Effects of skeleton photoperiods on melatonin secretion in the rainbow trout, Onchorhynchus mykiss (Walbaum). Aquacult. Fish. Manag. 24, 157–162. Arendt, J. (1985). Assay of melatonin and its metabolites: Results in normal and unusual environments. In ‘‘Proceedings of the First International Conference on Melatonin in Humans’’ (R. J. Wurtman and F. Waldhauser, Eds.), pp. 11–32. Armstrong, S. M., and Redman, J. R. (1993). Melatonin and circadian rhythmicity. In ‘‘Melatonin: Biosynthesis, Physiological Effects, and Clinical Applications’’ (H. S. Yu and R. J. Reiter, Eds.), pp. 187–224. Audet, C., Fitzgerald, G. J., and Guderley, H. (1986). Photoperiod effects on plasma cortisol levels in Gasterosteus aculeatus. Gen. Comp. Endocrinol. 61, 76–81. Begay, V., Falco´n, J., Cahill, G. M., Klein, D. C., and Coon, S. L. (1998). Transcripts endoding two melatonin synthesis enzymes in the teleost pineal organ: circadian regulation in pike and zebrafish, but not in trout. Endocrinology 139, 905–912.
Diel Rhythms of Serum Components in Dentex Bolliet, V., Ali, M. A., Lapointe, J. F., and Falcon, J. (1996). Rhythmic melatonin secretion in different teleost species: an in vitro study. J. Comp. Physiol. B 165, 677–683. Boujard, T., and Leatherland, J. F. (1992a). Circadian rhythms and feeding time in fishes. Environ. Biol. Fishes 35, 109–131. Boujard, T., and Leatherland, J. F. (1992b). Diel pattern of hepatosomatic index, liver glycogen and lipid content, plasma non esterified free fatty acid, glucose, T3, T4, growth hormone and cortisol concentrations in Oncorhynchus mykiss held in different photoperiod regimes and fed using demand-feeders. Fish Physiol. Biochem. 10, 111–122. Boujard, T., Brett, S., Lin, L., and Leatherland, J. F. (1993). Effect of restricted access to demand feeders on diurnal pattern of liver composition, plasma metabolites and hormone levels in Oncorhynchus mykiss. Fish Physiol. Biochem. 11, 337–344. Bromage, N. R., Randall, C. F., Porter, M. J. R., and Davies, B. (1995). How do photoperiod, the pineal gland, melatonin, and circannual rhythms interact to co-ordinate seasonal reproduction in salmonid fish? In ‘‘Proceedings of Fifth International Symposium on the Reproductive Physiology of Fish’’ (F. W. Goetz and P. Thomas, eds.), pp. 164–166. Fish Symposium 95, Austin. Carrillo, M., Zanuy, S., Montoro, R., and Ollevier, F. (1986). Daily patterns of blood electrolytes in sea bass adapted to different osmotic media at different times of the year. J. Interdisc. Cycle Res. 17, 69–80. Chan, D. K. O., and Woo, N. Y. S. (1978). Effect of cortisol on the metabolism of the eel, Anguilla japonica. Gen. Comp. Endocrinol. 35, 205–215. Delahunty, G., Bauer, G., Prack, M., and deVlaming, V. (1978). Effect of pinealectomy and melatonin treatment on liver and plasma metabolites in the goldfish, Carassius auratus. Gen. Comp. Endocrinol. 35, 99–109. Delahunty, G., Schreck, C. B., and deVlaming, V. L. (1977). Effect of pinealectomy, reproductive state, feeding regime and photoperiod on plasma cortisol in goldfish. Am. Zool. 17, 873. deVlaming, V. L., Sage, M., and Charlton, C. B. (1974a). The effects of melatonin treatment on gonadosomatic index in the teleost, Fundulus similis and the tree frog Hyla cinerea. Gen. Comp. Endocrinol. 22, 433–438. deVlaming, V. L., Sage, M., Charlton, C. B., and Tiegs, B. (1974b). The effects of melatonin on lipid deposition in Cyprinodontid fishes and on pituitary prolactin activity in Fundulus similis. J. Comp. Physiol. 94, 309–319. Falco´n, J., Brun-Marmillon, J., Claustrat, B., and Collin, J.-P. (1989). Regulation of melatonin secretion in a photoreceptive pineal organ: An in vitro study in the pike. J. Neurosci. 45, 479–486. Folmar, L. C., and Dickhoff, W. W. (1981). Evaluation of some physiological parameters as predictive indices of smoltification. Aquaculture 23, 309–324. Fraser, S., Cowen, P., Franklin, M., and Arendt, J. (1983). Direct radioimmunoassay for melatonin in plasma. Clin. Chem. 29, 396–397. Garcia, L. E., and Meier, A. H. (1973). Daily rhythms in concentration of plasma cortisol in male and female gulf killifish, Fundulus grandis. Biol. Bull. 144, 471–479. Gern, W. A., and Greenhouse, S. S. (1988). Examination of in vitro
249 melatonin secretion from superfused trout (Salmo gairdneri) pineal organs maintained under diel illumination or continuous darkness. Gen. Comp. Endocrinol. 71, 163–174. Gern, W. A., Owens, D. W., and Ralph, C. L. (1978). Plasma melatonin in the trout: Day-night change demonstrated by radioimmunoassay. Gen. Comp. Endocrinol. 34, 453–458. Gern, W. A., Gorell, T. A., and Owens, D. W. (1980). Melatonin and pigment cells rhythmicity. In ‘‘Melatonin: Current Status and Perspectives’’ (N. Birau and W. Schloot, Eds.), ‘‘Advances in Bioscience,’’ Vol. 29, pp. 223–233. Pergamon, Oxford. Gern, W., Dickhoff, W. W., and Folmar, L. C. (1984). Increases in plasma melatonin titers accompanying seawater adaptation of coho salmon (Oncorhynhus kisutch). Gen. Comp. Endocrinol. 55, 458–462. Henderson, I. W., and Garland, H. O. (1980). The interrenal gland in pisces. Part 2. Physiology. In ‘‘General, Comparative and Clinical Endocrinology of the Adrenal Cortex’’ (I. Chester Jones and I. W. Henderson, Eds.), Vol. 3, pp. 473–523. Academic Press, New York. Iigo, M., and Aida, K. (1995). Effects of season, temperature, and photoperiod on plasma melatonin rhythms in the goldfish, Carassius auratus. J. Pineal. Res. 18, 62–68. Iigo, M., Furukawa, K., Hattori, A., Ohtani-Kaneko, R., Hara, M., Suzuki, T., Tabata, M., and Aida, K. (1997). Ocular melatonin rhythms in the goldfish, Carassius auratus. J. Biol. Rhythms 12, 182–192. Kavaliers, M. (1980). The pineal organ and circadian rhythms of fishes. In ‘‘Environmental Physiology of Fishes’’ (M. A. Ali, Ed.), pp. 631–645. Plenum, New York. Kezuka, H., Furukawa, K., Aida, K., and Hanyu, I. (1988). Daily cucles in plasma melatonin levels under long or short photoperiod in the common carp, Cyprinus carpio. Gen. Comp. Endocrinol. 72, 296–302. Kezuka, H., Iigo, M., Furukawa, K., Aida, K., and Hanyu, I. (1992). Effects of photoperiod, pinealectomy and ophthalmectomy on circulating melatonin rhythms in the goldfish, Carassius auratus. Zool. Sci. 9, 1047–1053. Khan, I. A., and Joy, K. P. (1988). Diurnal variations in hypothalamic monoamine levels in the teleost Channa punctatus (Bloch) in response to melatonin under two photothermal conditions. Fish Physiol. Biochem. 5, 187–190. Kukkonen, S., and Laitinen, J. (1987). Melatoniinierityksen vuodenaikaiset muutokset kirjolohella. Suomen Ela¨inla¨a¨ka¨rilehti 93, 359–361. Kulczykowska, E. (1995). Arginine vasotocin—melatonin interactions in fish: A hypothesis. Rev. Fish Biol. Fish 5, 96–102. Ku¨hn, E. R., Corneillie, S., and Ollevier, F. (1986). Circadian variations in plasma osmolality, electrolytes, glucose, and cortisol in carp (Cyprinus carpio). Gen. Comp. Endocrinol. 61, 459–468. Laidley, C. W., and Leatherland, J. F. (1988). Circadian studies of plasma cortisol, thyroid hormone, protein, glucose and ion concentration, liver glycogen concentration and liver and spleen weight in rainbow trout, Salmo gairdneri Richardson. Comp. Biochem. Physiol. A 89, 495–503. Max, M., and Menaker, M. (1992). Regulation of melatonin production by light, darkness, and temperature in the trout pineal. J. Comp. Physiol. A 170, 479–489. Meier, A. H., and Fivizzani, A. J. (1975). Changes in the daily rhythm
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250 of plasma corticosterone concentration related to seasonal conditions in the white-throated sparrow, Zonotrichia albicollis. Proc. Soc. Exp. Biol. Med. 150, 356–362. Nichols, D. J., and Weisbart, M. (1984). Plasma cortisol concentrations in Atlantic Salmon, Salmo salar: Episodic variations, diurnal change, and short term response to adrenocorticotrophic hormone. Gen. Comp. Endocrinol. 56, 169–176. Pang, S.-F., Brown, G. M., Grota, L. J., Chambers, J. W., and Rodman, R. L. (1977). Determination of N-acetylserotonin and melatonin activities in the pineal gland, retina, Harderian gland, brain and serum of rat and chickens. Neuroendocrinology 23, 1–13. Pavlidis, M., Berry, M., Divanach, P., and Kentouri, M. (1997). Diel pattern of haematocrit, serum metabolites, osmotic pressure, electrolytes and thyroid hormones in sea bass and sea bream. Aquacult. Int. 5, 237–247. Peter, R. E., Hontela, A., Cook, A. F., and Paulencu, C. R. (1978). Daily cycles in serum cortisol levels in the goldfish: Effects of photoperiod, temperature, and sexual condition. Can. J. Zool. 56, 2443– 2448. Pickering, A. D., and Pottinger, T. G. (1983). Seasonal and diel changes in plasma cortisol levels of the brown trout, Salmo trutta L. Gen. Comp. Endocrinol. 49, 232–239. Rance, T. A., Baker, B. I., and Werbley, G. (1982). Variations in plasma cortisol concentrations over a 24-hour period in the rainbow trout Salmo gairdneri. Gen. Comp. Endocrinol. 48, 269–274. Randall, C. F., Trush, M., and Bromage, N. (1991). Absence of an endogenous component regulating melatonin secretion in the rainbow trout. In ‘‘Advances in Pineal Research: 5’’ (J. Arendt and P. Pe´vet, Eds.), pp. 279–281. Libbey, London. Randall, C. F., Bromage, N. R., Thorpe, J. E., Miles, M. S., and Muir, J. S. (1995). Melatonin rhythms in Atlantic salmon (Salmo salar) maintained under natural and out-of-phase photoperiods. Gen. Comp. Endocrinol. 98, 73–86. Redgate, E. S. (1974). Neural control of pituitary-adrenal activity in Cyprinus carpio. Gen. Comp. Endocrinol. 22, 35–41.
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Pavlidis et al. Reiter, R. (1991). Pineal melatonin: Cell biology of its synthesis and of its physiological interactions. Endocrine Rev. 11, 151–180. Rollag, M. D., and Niswender, G. D. (1976). Radioimmunoassay of serum concentrations of melatonin in sheep exposed to different lighting regimens. Endocrinology 98, 482–489. Scott, A. P., Mackenzie, D. N., and Stacey, N. E. (1984). Endocrine changes during natural spawning of the white sucker Catostomus commersoni. II. Steroid hormones. Gen. Comp. Endocrinol. 56, 349–359. Spieler, R. E., and Noeske, T. A. (1984). Effects of photoperiod and feeding schedule on diel variations of locomotor activity, cortisol, and thyroxine in goldfish. Trans. Am. Fish. Soc. 115, 528–539. Stokkan, K.-A., Vaughan, M. K., Reiter, R. J., Folkow, L. P., Mårtensson, P.-E., Sager, G., Lydersen, C., and Blix, A. S. (1995). Pineal and thyroid functions in newborn seals. Gen. Comp. Endocrinol. 98, 321–331. Thibault, C., Falcon, J., Greenhouse, S. S., Lowery, C. A., Gern, W. A., and Collin, J. P. (1993). Regulation of melatonin production by pineal photoreceptory cells: role of cyclic nucleotides in trout (Oncorhynhus mykiss). J. Neurochem. 61, 332–339. Valtonen, M., Laitinen, J. T., and Eriksson, L. (1993). Renal melatonin excretion in sheep is enhanced by water diuresis. J. Endocrinol. 138, 445–450. Underwood, H. (1985). Pineal melatonin rhythms in the lizard Anolis carolinensis: Effects of light and temperature cycles. J. Comp. Physiol. A 157, 57–65. Zachmann, A., Knijff, S. C. M., Ali, M. A., and Anctil, M. (1992a). Effects of photoperiod and different intensities of light exposure on melatonin levels in the blood, pineal organ, and retina of the brook trout (Salvelinus fontinalis Mitchill). Can. J. Zool. 70, 25–29. Zachmann, A., Falco´n, J., Knijff, S. C. M., Bolliet, V., and Ali, M. A. (1992b). Effects of photoperiod and temperature on rhythmic melatonin secretion from the pineal organ of the white sucker (Catostomus commersoni) in vitro. Gen. Comp. Endocrinol. 86, 26–33.
The Effect of Photoperiod on Diel Rhythms in Serum Melatonin, Cortisol, Glucose, and Electrolytes in the Common Dentex, Dentex dentex M. Pavlidis,*,1 L. Greenwood,† M. Paalavuo,‡ H. Mo¨lsa¨,‡ and J. T. Laitinen§ *Department of Aquaculture, Institute of Marine Biology of Crete, P.O. Box 2214, GR-710 03, Heraklio, Crete, Greece; †Lowestoft Laboratory, Centre for Environment, Fisheries, and Aquaculture Science, Lowestoft, Suffolk NR33 OHT, England; ‡Department of Applied Zoology and Veterinary Medicine, University of Kuopio, P.O. Box 1627, FIN-702 11, Kuopio, Finland; and §Department of Physiology, University of Kuopio, P.O. Box 1627, FIN-702 11, Kuopio, Finland Accepted September 25, 1998
first part of the scotophase under all regimes, except the 16L:8D where no diel rhythmicity was detected. During the photophase, cortisol was positively correlated with glucose, Naⴙ, and Clⴚ and negatively with Kⴙ. During the scotophase, melatonin was positively correlated with glucose and electrolytes. Results indicated that cortisol may be responsible for the observed rhythmicity of glucose and that melatonin may play a role in glucose and ion regulation in common dentex. r 1999 Academic Press Key Words: diel rhythms; cortisol; common dentex (Dentex dentex); electrolytes; glucose; melatonin; photoperiod.
Diel rhythms in serum concentrations of melatonin, cortisol, glucose, sodium, chloride, and potassium were studied in the common dentex, Dentex dentex, under different photoperiods (DD, 8L:16D, 12L:12D, 16L:8D). Photoperiod affected both the diel rhythms and the absolute values of the estimated blood components. Regardless of the photoperiod, melatonin titers were elevated during the scotophase (384.3 ⴞ 13.9 pg/ml) compared with a mean baseline level of 54.4 ⴞ 2.7 pg/ml during the photophase. Serum melatonin concentrations reflected the prevailing photoperiod and constantly elevated melatonin levels with no diel rhythmicity were evident in fish held in the DD protocol. A circadian-like pattern in serum cortisol was observed in fish that were kept at the DD and 8L:16D protocols with cortisol peak at 18:00 h in the night. Fish exposed to the 16L:8D regime showed highest cortisol levels at 10:00 h, while no rhythmicity was evident under the 12L:12D protocol. A phase shift of 4 h between the peaks of cortisol and glucose was evident in fish exposed to the DD, 8L:16D, and 12L:12D regimes. Diel patterns of changes in serum Naⴙ and Clⴚ were observed only in the fish held in the DD protocol. Serum Kⴙ values were lowest during the
All vertebrates display a similar diel pattern of changes in plasma melatonin levels in response to conventional light-dark cycles, with baseline levels during the photophase and high concentrations during the scotophase (Rollag and Niswender, 1976; Pang et al., 1977; Gern et al., 1978; Underwood, 1985; Armstrong and Redman, 1993; Stokkan et al., 1995). In addition, the role of melatonin in conveying information on the diurnal and seasonal changes in daylength has been reported in various fish species: the rainbow trout, Oncorhynchus mykiss (Gern et al., 1978; Randall et al., 1991; Alvarin˜o et al., 1993); brook trout, Salvelinus fontinalis (Zachmann et al., 1992a); Atlantic salmon, Salmo salar (Randall et al., 1995); common carp, Cypri-
1 To whom correspondence should be addressed at Department of Aquaculture, Institute of Marine Biology of Crete, P.O. Box 2214, GR-710 03, Heraklion, Crete, Greece. Fax: 081-241882. E-mail: [email protected].
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Diel Rhythms of Serum Components in Dentex
nus carpio (Kezuka et al., 1988); pike, Esox lucius (Falco´n et al., 1989); goldfish, Carassius auratus (Kezuka et al., 1992); white sucker, Catostomus commersoni (Zachmann et al., 1992b); and catfish, Silurus asotus (Iigo et al., 1997). Although the role of melatonin in transducing the information about ambient photoperiodic changes into rhythmic bodily function such as circadian and seasonal rhythms and in mediating several light-cued physiological processes such as locomotory activity (Kavaliers, 1980) and pigmentation (Gern et al., 1980) is well documented, the action of melatonin in regulating metabolic and/or endocrine function in fish is unclear. Melatonin treatment significantly reduces the hypothalamic noradrenaline content in Chana punctatus (Khan and Joy, 1988), is hypoglycemic in sham-operated but not pinealectomized goldfish, and increases liver glycogen levels in sham-operated but not pinealectomized goldfish, C. auratus (Delahunty et al., 1978). Interactions among cortisol, arginine vasotocin (hormones closely associated with electrolyte balance), and melatonin have been reported in teleosts (Delahunty et al., 1977; Kulczykowska, 1995). In addition, a similar pattern of changes in plasma titers of melatonin, sodium, and chloride during seawater adaptation of coho salmon has been reported by Folmar and Dickoff (1981), indicating a possible role of melatonin in osmoregulation and/or stress adaptation in seawater. Data on stenohaline marine species are lacking. The common dentex, Dentex dentex (L., 1758) is a common marine fish whose melatonin secretory profiles and diel rhythms of serum components are unknown. The objectives of the present study were to (1) describe the daily melatonin rhythms in the blood of the D. dentex maintained under different photoperiodic regimes; and (2) examine possible relationships between melatonin rhythms and other serum components (cortisol, glucose, sodium, potassium, chloride) implicated in osmoregulation in this stenohaline marine species.
MATERIALS AND METHODS Animals One hundred twenty immature 9-month-old common dentex weighing 140.2 ⫾ 8.2 g, reared under
ambient photothermal conditions at the Aquaculture Research Station of the IMBC, were transferred to indoor laboratory facilities, in February 1996. Fish were distributed randomly into two 6 ⫻ 500 L circular tanks (10 fish/tank) supplied with continuously aerated and running seawater (water flow, 8–10 L/min; dissolved O2, 4.5–6.0 mg/L, salinity, 38–40%). Fish were fed once a day by hand with wet pellets (50% raw fish, 50% commercial pellets). On the day of sampling no food was given. Black plastic curtains surrounded each set of tanks and artificial illumination was achieved using fluorescent strip lights located 130 cm above the water surface, controlled by electric time clocks with no twilight period. Light intensity was 150 lx at the water surface (tank depth: 1 m).
Experimental Design Fish were acclimated to each respective photoperiod treatment for 5–6 weeks prior to sampling. In all experiments dawn was set at 06:00 h. Sampling began at 06:00 h and was repeated at 4-h intervals (06:00, 10:00, 14:00, 18:00, 22:00, 02:00 h) over a 24-h cycle. Ten fish were used at each time interval. To minimize stress, 10 min prior to sampling the level of the water was reduced (by siphoning) and anesthetic (0.25 ml/L Ethylene glycol monophenyl ether, Merck, Hohenbrunn) was added to the tank water. Lightly anesthetized fish were netted, their head was covered with a towel, and blood was withdrawn from the caudal vessel. Clotted blood was centrifuged at 2500g and serum was collected, aliquoted for the different assays, and stored at ⫺20°C until analysis. Sampling took 15–20 min per tank. When blood collection was completed fish were placed in 70-L tanks provided with oxygen for recovery and then were returned to the experimental tanks. Two series of experiment were performed: A. In the first, fish (n ⫽ 60) were exposed to 12L:12D light-dark cycle (water temperature 15.6 ⫾ 0.3°C, sampling on April 2) and then subjected to 16L:8D regime (17.0 ⫾ 0.8°C, sampling on May 10). B. In the second, fish (n ⫽ 60) were subjected to 8L:16D light-dark regime (sampling on April 2) and then placed on constant darkness (DD) (sampling on May 10).
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Pavlidis et al.
say of diethyl ether-extracted serum samples as described by Scott et al., (1984). Briefly, serum aliquots were shaken with 4 ml of diethyl ether. The serum was frozen in liquid nitrogen and the ether decanted into a 12 ⫻ 75-mm borosilicate glass tube. The ether was then evaporated and the residue containing free cortisol redissolved in assay buffer. Aliquots of 50 or 100 µl of these diluted samples were used in the radioimmunoassay. The cortisol antibody was purchased from Scottish Antibody Production Unit (Strathclyde, Scotland) and tritiated cortisol purchased from Amersham International Ltd. (UK). The inter- and intraassay coefficients of variation were 5.1% (n ⫽ 14) and 1.4% (n ⫽ 20), respectively. Glucose was estimated by the enzymatic colorimetric (GOD/PAP) method (Biosis, Greece). Serum electrolytes were determined by the use of ion-selective electrodes (Ciba Corning 644 Na⫹/K⫹/Cl⫺ analyzer).
Analytical Techniques Melatonin was analyzed from chloroform-extracted serum samples using a specific RIA (Fraser et al., 1983; Valtonen et al., 1993). Briefly, duplicate samples of serum, melatonin standards (5–200 pg/ml), and lowand high-melatonin control samples (11 and 66 pg/ml, respectively) in a total volume of 0.5 ml were extracted into 3.0 ml of chloroform. The dried residue was reconstituted in phosphate-buffered saline, pH 7.0, containing 1 g of gelatin per liter and assayed for melatonin using the G/S/704-8483 antiserum (Stockgrand Ltd, University of Surrey, Guildford, Surrey UK) and tritiated melatonin as a tracer. Displacement curves (three different pools of dentex serum, one daytime pool and two nighttime pools, with three different serum volumes, day pools 25, 50, and 100 µl; night pools 10, 25, and 50 µl) demonstrated linear parallelism after logit-log transformation (standard curve, r2 ⫽ 1.000, slope ⫽ ⫺0.995; day pool, r2 ⫽ 1.000, slope ⫽ ⫺0.952; night pool 1, r2 ⫽ 0.99, slope ⫽ ⫺0.943; night pool 2, r2 ⫽ 0.999, slope ⫽ ⫺0.942). The recovery of melatonin (10, 20, or 50 pg) added to dentex serum was 96.1 ⫾ 3.0% (mean ⫾ SEM, n ⫽ 3). The intraassay coefficients of variation were 6.5 and 3.1% for melatonin concentrations of 11 and 66 pg/ml, respectively (n ⫽ 6). The interassay coefficients of variation for these melatonin concentrations were 11.6 and 5.2%, respectively. Cortisol levels were determined by radioimmunoas-
Statistical Analysis Data are expressed as means ⫾ standard error. However, for the melatonin data of Table 1, the mean, standard deviation, range, and intragroup coefficient of variation (CV%) are presented to illustrate the fact that not a single individual fish in the DD group exhibited low daytime serum melatonin concentrations at any time point studied. The KolomogorovSmirnov test was applied to check for a normally
TABLE 1 Dentex Serum Melatonin Concentrations (pg/ml), Mean, SD, Range, and CV at Each Sampling Point and Photoperiod Regime 12L:12D
Hour of sampling
Mean
SD
06:00 10:00 14:00 18:00 22:00 02:00
58.0 68.7 35.3 267.2 282.3 224.5
21.9 31.3 13.7 91.7 83.7 52.2
06:00 10:00 14:00 18:00 22:00 02:00
50.9 52.6 226.7 330.5 327.5 247.8
19.0 28.1 129.8 123.0 65.5 64.0
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Range 39–106 40–154 21–54 155–480 145–385 132–280 8L:16D 29–87 19–101 81–403 188–596 269–459 154–338
16L:8D CV (%)
Mean
SD
Range
CV (%)
38 45 39 34 30 23
64.8 66.9 46.5 44.0 329.6 484.1
30.6 29.2 12.3 6.9 138.6 101.1
20–95 30–134 29–69 31–53 106–570 311–685
47 44 26 16 42 21
37 53 57 37 20 26
458.7 508.0 452.7 427.4 604.9 579.1
90.6 150.0 98.7 95.6 176.9 168.5
329–630 234–797 354–641 274–627 337–826 304–869
20 29 22 22 29 29
DD
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Diel Rhythms of Serum Components in Dentex
distributed population and the Bartlett test was applied to verify the homogeneity of variances. One-way analysis of variance (ANOVA) or the Kruskal-Wallis test (when variances were not homogeneous) was used to test for significant changes between sampling times within a single photoperiod or between the different photoperiod regimes within a single serum component. If significant (P ⬍ 0.05), Tukey’s significant means test or Dunn’s multiply comparison test was applied to compare mean values at different sampling times. Comparison of the estimated serum parameters between the photo- and scotophase within each respective photoperiod protocol was performed by the t test or the Mann-Whitney rank sum test. Pearson product moment correlation was applied to measure the strength of association between pairs of the estimated variables.
fish held in the other experimental photoperiods (Table 1). Moreover, the intragroup coefficient of variation was similar between each group, indicating that melatonin concentrations in the DD group did not deviate from the mean more than individual values in other groups and time-points; this is in contrast to the situation where individual fish would exhibit nonsynchronous free-running melatonin rhythm with low daytime levels randomly found at each time point. Fish under the 8L:16D, 12L:12D, and 16L:8D photoperiods displayed a similar diel pattern of melatonin profile; minimum values occurred 4 h prior to lights off and maximum values within 0–4 h after lights off (Fig. 2). As the duration of photophase increased, the duration of elevated melatonin levels decreased, being shortest in fish held under the 16L:8D protocol. There was no significant effect of the order of sampling within a tank.
RESULTS
Cortisol
Melatonin Light significantly affected both the absolute values and the diel rhythms of serum melatonin concentrations. Regardless of the photoperiod regimen, melatonin levels were significantly higher during the scotophase (384.3 ⫾ 13.9 pg/ml) and returned to a mean baseline level of 54.4 ⫾ 2.7 pg/ml during the photophase (Fig. 1). No diel rhythm in serum melatonin was evident in fish held in the DD protocol. In particular, individual melatonin concentration at each sampling was significantly higher than the respective values in
FIG. 1. Serum melatonin (means ⫾ SEM) in the common dentex, Dentex dentex, during the photo- and scotophase of the experimental photoperiod protocols.
Significant fluctuations in serum cortisol were observed in fish under the 16L:8D, 8L:16D, and DD protocols, but not in the 12L:12D protocol (Fig. 3). Fish exposed to the long photoperiod (16L:8D) had the lowest cortisol levels in the middle of the dark period (02:00 h) and the highest levels 4 h after the lights on. In constant darkness (DD), serum cortisol levels were at a minimum between 02:00 and 06:00 h and at a maximum at 18:00 h. The fish on short photoperiod (8L:16D) displayed minimum cortisol values in the middle of the photophase (10:00 h) and a maximum between 18:00 and 22:00 h, 4 to 8 h after lights off. In the 12L:12D photoperiod, serum cortisol levels were relatively high at all time points examined with the exception of low levels at 06:00 h, but no significant rhythmicity was apparent. Regardless of photoperiod, there was a time lag of 8 to 12 h between the minimum and the maximum in cortisol level. There was no statistically significant difference in the absolute cortisol levels in fish held under the different photoperiod protocols (Table 2). However, in fish exposed to the 8L:16D and 16L:8D regimes, differences in the mean cortisol concentrations between the photo- and scotophase were observed; in the 8L:16D regime lower cortisol levels were found during the photophase (11.3 ⫾ 2.0 ng/ml) and higher during the scotophase (21.6 ⫾ 3.1 ng/ml), while in the 16L:8D there was an
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opposite pattern (photophase, 28.2 ⫾ 3.9 ng/ml; scotophase, 14.7 ⫾ 3.6 ng/ml). No significant effect of the order of sampling within a tank was observed.
Glucose There were diel rhythms in serum glucose levels in fish held in the DD, 8L:16D, and 12L:12D light-dark
FIG. 3. Photoperiod effect on serum cortisol levels in the common dentex, Dentex dentex, sampled at 4-h periods throughout the day. For details see legend of Fig. 2.
FIG. 2. Photoperiod effect on serum melatonin levels in the common dentex, Dentex dentex, sampled at 4-h periods throughout the day. Data are means ⫾ SEM (n ⫽ 10/sampling point). Dark horizontal lines indicate period of darkness (✬, lights-on; 䊉, lightsoff). Means with different letters differ significantly from one another, P ⬍ 0.05).
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cycles (Fig. 4). The lowest glucose levels occurred at 10:00 h for the DD and 12L:12D protocols or at 14:00 h for the 8L:16D protocol, and serum glucose concentrations attained maximum values during the scotophase (22:00 h). Higher absolute glucose concentrations were found in fish exposed to constant darkness (DD) than in fish held in the 8L:16D protocol (Table 2). No differences in the mean glucose levels between the photo- and scotophase were detected. An effect of the order of sampling with an increase in plasma glucose values with increasing order of sample from each tank
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Diel Rhythms of Serum Components in Dentex
TABLE 2 Absolute Values of Serum Parameters (Mean ⫾ SEM) in Dentex dentex Acclimated to Different Photoperiods Group
T (°C)
Cortisol (ng/ml)
Glucose (mmol/L)
Na⫹ (mmol/L)
K⫹ (mmol/L)
Cl⫺ (mmol/L)
DD 8L:16D 12L:12D 16L:8D
17.0 ⫾ 0.8 15.6 ⫾ 0.3 15.6 ⫾ 0.3 17.0 ⫾ 0.8
18.3 ⫾ 3.2 18.8 ⫾ 2.2 19.6 ⫾ 1.9 22.6 ⫾ 2.9
4.55 ⫾ 0.27a 3.54 ⫾ 0.16b 3.65 ⫾ 0.19 3.92 ⫾ 0.18
203.0 ⫾ 1.0a 196.8 ⫾ 0.6bc 199.1 ⫾ 0.7b 200.6 ⫾ 0.8a
2.94 ⫾ 0.07a 2.62 ⫾ 0.06c 2.51 ⫾ 0.06c 2.20 ⫾ 0.06b
186.2 ⫾ 0.8a 177.7 ⫾ 0.5c 178.4 ⫾ 0.7c 182.2 ⫾ 0.8b
Note. Means with different letters differ significantly from one another a–b, c–d (P ⬍ 0.05).
was observed in all protocols except for the 16L:8D regime.
Electrolytes Statistically significant changes in mean serum Na⫹ and Cl⫺ levels were seen only in fish exposed to constant darkness, in which maximum ion concentrations were observed at 22:00 h (Fig. 5). Under the other photoperiods no diel rhythmicity in Na⫹ and Cl⫺ was detected. There were higher absolute values for Na⫹ and Cl⫺ in fish exposed to the DD and 16L:8D photoperiods compared with those held under 8L:16D and 12L:12D protocols (Table 2). Significant diel patterns of serum K⫹ levels were found under all photoperiodic conditions, with the exception of the 16L:8D protocol. Fish held in the 8L:16D and 16L:8D protocols showed minimum potassium values at 18:00 h, while fish exposed to the DD regime exhibited a minimum at 06:00 h (Fig. 6). Higher absolute potassium values were found in fish held in the DD protocol whereas there were lower values in fish exposed to the 16L:8D photoperiod (Table 2). There were no differences in the mean values of the estimated serum electrolytes between the light and dark period of each treatment.
Correlation between the Serum Parameters In fish under the 8L:16D protocol, melatonin was positively correlated with cortisol (r ⫽ 0.523,P ⬍ 0.001), while in the 16L:8D regime a negative correlation was found (r ⫽ ⫺0.281, P ⬍ 0.05). When the photophase of all regimes was analyzed the following relationships were found: cortisol was positively correlated with glucose (r ⫽ 0.423, P ⬍ 0.001), Na⫹ (r ⫽ 0.341, P ⬍ 0.01), and Cl⫺ (r ⫽ 0.364, P ⬍ 0.001) and negatively with K⫹ (r ⫽ ⫺0.248, P ⬍ 0.05); glucose with Na⫹ (r ⫽ 0.308,
P ⬍ 0.01) and Cl⫺ (r ⫽ 0.313, P ⬍ 0.01). When the scotophase was analyzed significant positive correlations were observed between melatonin and glucose (r ⫽ 0.307, P ⬍ 0.001), Na⫹ (r ⫽ 0.353, P ⬍ 0.001), K⫹ (r ⫽ 0.221, P ⬍ 0.01), and Cl⫺ (r ⫽ 0.475, P ⬍ 0.001); glucose and Na⫹ (r ⫽ 0.411, P ⬍ 0.001), K⫹ (r ⫽ 0.348, P ⬍ 0.001), and Cl⫺ (r ⫽ 0.378, P ⬍ 0.001).
DISCUSSION The present study was conducted to gain basic information on the effects of different photoperiods on certain serum characteristics of the common dentex. This is the first study to document diel changes in serum melatonin, cortisol, glucose, and electrolytes concentrations in this fish. The experimental design aimed to overcome possible effects of stocking density, feeding regime, size, and sexual condition on the diel pattern of the estimated parameters. The validation experiments indicated that melatonin levels were accurately and quantitatively estimated from chloroform-extracted serum samples. In most mammals, daytime serum melatonin levels are typically ⬍10 pg/ml and during the hours of darkness, 50 and 150 pg/ml (Arendt, 1985). In the fish studied so far, circulating melatonin levels are clearly higher between 50 and 100 pg/ml during the photophase for the pike (Falco´n et al., 1989), the rainbow trout (Gern et al., 1978; Kukkonen and Laitinen, 1987), the Atlantic salmon (Bromage et al., 1995), and the common carp (Kezuka et al., 1988). In these studies, nightime melatonin levels typically fluctuated around 300–500 pg/ml with somewhat lower concentrations in some of the studies with rainbow trout. Thus the melatonin levels currently reported for the dentex closely resemble
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Pavlidis et al.
those found in other species of fish. A recent study revealed, however, that in the catfish on a 12L:12D photoperiod, maximal nocturnal plasma melatonin levels were only about 40 pg/ml (Iigo et al., 1997) suggesting that in fish, there may be marked speciesspecific differences in the diurnal rhythm of circulating melatonin concentration. Although photoperiod appears to be the major regulator of melatonin secretion in many warmblooded animals (Reiter, 1991), a significant effect of environmental temperature on the amplitude of mela-
FIG. 5. Photoperiod effect on serum sodium and chloride levels in the common dentex, Dentex dentex, sampled at 4-h periods throughout the day. For details see legend of Fig. 2.
FIG. 4. Photoperiod effect on serum glucose levels in the common dentex, Dentex dentex, sampled at 4-h periods throughout the day. For details see legend of Fig. 2.
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tonin rhythm has been clearly documented in poikilotherms (Underwood, 1985; Iigo and Aida, 1995; Randall et al., 1995). In the present study, peak melatonin levels were around 300 pg/ml in the two photoperiodic groups sampled in April (8L:16D and 12L:12D; water temperature, 15.6 ⫾ 0.3°C) and around 500 pg/ml when the same individuals were sampled again for the two other photoperiods in May (DD and 16L:8D; water temperature, 17.0 ⫾ 0.8°C). Although the temperature change between these two sampling sessions was relatively modest, these observations could reflect a temperature-dependent increase in the amplitude of melatonin secretion rhythm in the dentex. The present data demonstrate that plasma melatonin concentrations provide an accurate representation of the prevailing photoperiod in the dentex, suggesting that photoperiodic manipulations will result in
Diel Rhythms of Serum Components in Dentex
FIG. 6. Photoperiod effect on serum potassium levels in the common dentex, Dentex dentex, sampled at 4-h periods throughout the day. For details see legend of Fig. 2.
highly predictable changes in the duration of nocturnal melatonin signal in this fish species; this is similar to observations on other fish species (Iigo and Aida, 1995; Randall et al., 1995). Moreover, constantly elevated melatonin levels were evident in dentex held in DD, suggesting that in contrast to most teleosts, in which melatonin production is driven by a pineal clock, melatonin production in the dentex occurs primarly in response to darkness, as has been described in the trout (Gern and Greenhouse, 1988;
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Randall et al., 1991; Max and Menaker, 1992; Thibault et al., 1993; Bolliet et al., 1996; Begay et al., 1998). Diel rhythmicity of circulating cortisol has been extensively studied in freshwater fish species (Boujard and Leatherland, 1992a). However, the exact pattern of cortisol rhythm varies, with peak values occurring during the daylight hours (Garcia and Meier, 1973; Spieler and Noeske, 1984; Boujard and Leatherland, 1992b), during nighttime (Redgate, 1974; Rance et al., 1982; Pickering and Pottinger, 1983), or during both periods (Peter et al., 1978; Ku¨hn et al., 1986). A circadianlike pattern in circulating cortisol was observed in common dentex that were exposed to a short photoperiod or constant darkness with a cortisol acrophase at 18:00 h. A phase shift of the cortisol peak was evident in fish exposed to long photoperiods, with an acrophase at 10:00 h in the morning, in a phase with a period of hyperphagia. Shifts in acrophases of cortisol rhythms have been described for several other animals (Meier and Fivizzani, 1975; Pickering and Pottinger, 1983; Ku¨hn et al., 1986), possibly reflecting differences in light/dark cycle, temperature, or feeding activity. No diel rhythm in cortisol was detected in fish held in the 12L:12D regime. This could be partly explained by the high individual variability in serum cortisol levels or by the fact that the 4-h interval between samplings could have masked a brief peak. Similar high variability among ‘‘unstressed’’ individuals or episodic elevation of circulating cortisol has been reported in fish (Pickering and Pottinger, 1983; Nichols and Weisbart, 1984; Audet et al., 1986). Serum glucose levels peaked during the scotophase (22:00 h) in all regimes apart from the 16L:8D in which there was no diel glucose rhythm. Similar results, with glucose acrophase during the scotophase, have been reported in sea bream and sea bass (Pavlidis et al., 1997), red porgy (M. Pavlidis, M. Paspatis, M. Koistinen, T. Paavola, P. Divanach, and M. Kentouri, unpublished data), rainbow trout (Boujard and Leatherland, 1992a; Boujard et al., 1993), and common carp (Ku¨hn et al., 1986). A phase shift of 4 h in acrophase of cortisol and glucose was evident in fish exposed to the DD, 8L:16D, and 12L:12D regimes. Therefore, these results indicate that the serum glucose zenith in the common dentex does not reflect food absorption from the gut, but may rather reflect gluconeogenic activity due to elevated cortisol concentrations. An effect of sampling sequence
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with increased serum glucose levels with passage of time was evident in all experiments. Laidley and Leatherland (1988) reported similar results in a study in rainbow trout. This difference does not nullify the observed glucose fluctuations. It should, however, be taken into consideration when planning for future experiments monitoring serum glucose in this particular species. Diel changes in serum sodium and chloride were observed only in fish on the DD protocol. Lowest potassium values were found during the first part of the scotophase in all regimes, except the 16L:8D in which there was no diel rhythmicity. Seasonal or diel variations in monovalent ions, although not necessarily nor consistently circadian in periodicity, have been reported for sea bream (Pavlidis et al., 1997), sea bass (Carillo et al., 1986; Pavlidis et al., 1997), rainbow trout (Laidley and Leatherland, 1988), and common carp (Ku¨hn et al., 1986). These changes may reflect circadian variations in ion exchanges across the gills and/or urinary electrolyte excretion. However, the biological significance, if any, of the observed diel K⫹ fluctuations remains to be elucidated. Interestingly, higher absolute values of Na⫹ and Cl⫺ paralleled the high melatonin titers in the DD and 16L:8D regimes, possibly reflecting temperature-dependent increases in the amplitude of electrolyte rhythm in the dentex. Remarkable, photoperiod-related, relationships between the estimated parameters were noted in the common dentex. Glucose was positively correlated with electrolytes, indicating a close relation between glucose and ion regulation. More importantly, cortisol was positively correlated during the photophase with glucose, Na⫹, and Cl⫺ and negatively with K⫹, supporting the proposed role of cortisol in intermediary metabolism and osmoregulation in fish (Henderson and Garland, 1980). The absence of such a correlation at the scotophase may explain the lack of correlation in plasma cortisol and glucose values reported in other studies (Chan and Woo, 1978; Ku¨hn, et al., 1986; Laidley and Leatherland, 1988). Melatonin was positively correlated with glucose and electrolytes during the scotophase in all groups. In addition, melatonin was positively correlated with cortisol in the 8L:16D protocol, and negatively in the 16L:8D regime, indicating a possible role of melatonin in electrolyte balance and metabolic processes as a result of a differential
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Pavlidis et al.
response to light. Cortisol and prolactin are known to either influence melatonin production or be affected by melatonin in fish (Delahunty et al., 1977; deVlaming et al., 1974a,b; Gern et al., 1984). Folmar and Dickhoff (1981) also reported a relationship between the highest serum concentrations of melatonin and Na⫹ ⫺ Cl⫺ during seawater adaptation of coho salmon. Whether this correlation indicates a direct or indirect (through other endocrine regulatory systems, e.g., argininvasotocin) involvement of melatonin in ion regulation is still unknown.
ACKNOWLEDGMENTS The study has been carried out with financial support from the Commission of the European Communities, Agriculture and Fisheries (FAIR) specific RTD programme, CT-95-0407, ‘‘Common dentex, a prime new species for aquaculture. Development of methods for reliable egg production.’’ It does not necessarily reflect its views and in no way anticipates the Commission’s future policy in this area. We are indebted to Prof. M. Kentouri for helpful comments on the manuscript. The authors express their gratitude to Mrs. M. Symsiridou for valuable efforts in fish husbandry and technical help, to Mr. S. Kukkonen for technical assistance in melatonin determination, and to the staff of Aquaculture Department of IMBC for their collaboration in carrying out this study.
REFERENCES Alvarin˜o, J. M. R., Randall, C. F., and Bromage, N. R. (1993). Effects of skeleton photoperiods on melatonin secretion in the rainbow trout, Onchorhynchus mykiss (Walbaum). Aquacult. Fish. Manag. 24, 157–162. Arendt, J. (1985). Assay of melatonin and its metabolites: Results in normal and unusual environments. In ‘‘Proceedings of the First International Conference on Melatonin in Humans’’ (R. J. Wurtman and F. Waldhauser, Eds.), pp. 11–32. Armstrong, S. M., and Redman, J. R. (1993). Melatonin and circadian rhythmicity. In ‘‘Melatonin: Biosynthesis, Physiological Effects, and Clinical Applications’’ (H. S. Yu and R. J. Reiter, Eds.), pp. 187–224. Audet, C., Fitzgerald, G. J., and Guderley, H. (1986). Photoperiod effects on plasma cortisol levels in Gasterosteus aculeatus. Gen. Comp. Endocrinol. 61, 76–81. Begay, V., Falco´n, J., Cahill, G. M., Klein, D. C., and Coon, S. L. (1998). Transcripts endoding two melatonin synthesis enzymes in the teleost pineal organ: circadian regulation in pike and zebrafish, but not in trout. Endocrinology 139, 905–912.
Diel Rhythms of Serum Components in Dentex Bolliet, V., Ali, M. A., Lapointe, J. F., and Falcon, J. (1996). Rhythmic melatonin secretion in different teleost species: an in vitro study. J. Comp. Physiol. B 165, 677–683. Boujard, T., and Leatherland, J. F. (1992a). Circadian rhythms and feeding time in fishes. Environ. Biol. Fishes 35, 109–131. Boujard, T., and Leatherland, J. F. (1992b). Diel pattern of hepatosomatic index, liver glycogen and lipid content, plasma non esterified free fatty acid, glucose, T3, T4, growth hormone and cortisol concentrations in Oncorhynchus mykiss held in different photoperiod regimes and fed using demand-feeders. Fish Physiol. Biochem. 10, 111–122. Boujard, T., Brett, S., Lin, L., and Leatherland, J. F. (1993). Effect of restricted access to demand feeders on diurnal pattern of liver composition, plasma metabolites and hormone levels in Oncorhynchus mykiss. Fish Physiol. Biochem. 11, 337–344. Bromage, N. R., Randall, C. F., Porter, M. J. R., and Davies, B. (1995). How do photoperiod, the pineal gland, melatonin, and circannual rhythms interact to co-ordinate seasonal reproduction in salmonid fish? In ‘‘Proceedings of Fifth International Symposium on the Reproductive Physiology of Fish’’ (F. W. Goetz and P. Thomas, eds.), pp. 164–166. Fish Symposium 95, Austin. Carrillo, M., Zanuy, S., Montoro, R., and Ollevier, F. (1986). Daily patterns of blood electrolytes in sea bass adapted to different osmotic media at different times of the year. J. Interdisc. Cycle Res. 17, 69–80. Chan, D. K. O., and Woo, N. Y. S. (1978). Effect of cortisol on the metabolism of the eel, Anguilla japonica. Gen. Comp. Endocrinol. 35, 205–215. Delahunty, G., Bauer, G., Prack, M., and deVlaming, V. (1978). Effect of pinealectomy and melatonin treatment on liver and plasma metabolites in the goldfish, Carassius auratus. Gen. Comp. Endocrinol. 35, 99–109. Delahunty, G., Schreck, C. B., and deVlaming, V. L. (1977). Effect of pinealectomy, reproductive state, feeding regime and photoperiod on plasma cortisol in goldfish. Am. Zool. 17, 873. deVlaming, V. L., Sage, M., and Charlton, C. B. (1974a). The effects of melatonin treatment on gonadosomatic index in the teleost, Fundulus similis and the tree frog Hyla cinerea. Gen. Comp. Endocrinol. 22, 433–438. deVlaming, V. L., Sage, M., Charlton, C. B., and Tiegs, B. (1974b). The effects of melatonin on lipid deposition in Cyprinodontid fishes and on pituitary prolactin activity in Fundulus similis. J. Comp. Physiol. 94, 309–319. Falco´n, J., Brun-Marmillon, J., Claustrat, B., and Collin, J.-P. (1989). Regulation of melatonin secretion in a photoreceptive pineal organ: An in vitro study in the pike. J. Neurosci. 45, 479–486. Folmar, L. C., and Dickhoff, W. W. (1981). Evaluation of some physiological parameters as predictive indices of smoltification. Aquaculture 23, 309–324. Fraser, S., Cowen, P., Franklin, M., and Arendt, J. (1983). Direct radioimmunoassay for melatonin in plasma. Clin. Chem. 29, 396–397. Garcia, L. E., and Meier, A. H. (1973). Daily rhythms in concentration of plasma cortisol in male and female gulf killifish, Fundulus grandis. Biol. Bull. 144, 471–479. Gern, W. A., and Greenhouse, S. S. (1988). Examination of in vitro
249 melatonin secretion from superfused trout (Salmo gairdneri) pineal organs maintained under diel illumination or continuous darkness. Gen. Comp. Endocrinol. 71, 163–174. Gern, W. A., Owens, D. W., and Ralph, C. L. (1978). Plasma melatonin in the trout: Day-night change demonstrated by radioimmunoassay. Gen. Comp. Endocrinol. 34, 453–458. Gern, W. A., Gorell, T. A., and Owens, D. W. (1980). Melatonin and pigment cells rhythmicity. In ‘‘Melatonin: Current Status and Perspectives’’ (N. Birau and W. Schloot, Eds.), ‘‘Advances in Bioscience,’’ Vol. 29, pp. 223–233. Pergamon, Oxford. Gern, W., Dickhoff, W. W., and Folmar, L. C. (1984). Increases in plasma melatonin titers accompanying seawater adaptation of coho salmon (Oncorhynhus kisutch). Gen. Comp. Endocrinol. 55, 458–462. Henderson, I. W., and Garland, H. O. (1980). The interrenal gland in pisces. Part 2. Physiology. In ‘‘General, Comparative and Clinical Endocrinology of the Adrenal Cortex’’ (I. Chester Jones and I. W. Henderson, Eds.), Vol. 3, pp. 473–523. Academic Press, New York. Iigo, M., and Aida, K. (1995). Effects of season, temperature, and photoperiod on plasma melatonin rhythms in the goldfish, Carassius auratus. J. Pineal. Res. 18, 62–68. Iigo, M., Furukawa, K., Hattori, A., Ohtani-Kaneko, R., Hara, M., Suzuki, T., Tabata, M., and Aida, K. (1997). Ocular melatonin rhythms in the goldfish, Carassius auratus. J. Biol. Rhythms 12, 182–192. Kavaliers, M. (1980). The pineal organ and circadian rhythms of fishes. In ‘‘Environmental Physiology of Fishes’’ (M. A. Ali, Ed.), pp. 631–645. Plenum, New York. Kezuka, H., Furukawa, K., Aida, K., and Hanyu, I. (1988). Daily cucles in plasma melatonin levels under long or short photoperiod in the common carp, Cyprinus carpio. Gen. Comp. Endocrinol. 72, 296–302. Kezuka, H., Iigo, M., Furukawa, K., Aida, K., and Hanyu, I. (1992). Effects of photoperiod, pinealectomy and ophthalmectomy on circulating melatonin rhythms in the goldfish, Carassius auratus. Zool. Sci. 9, 1047–1053. Khan, I. A., and Joy, K. P. (1988). Diurnal variations in hypothalamic monoamine levels in the teleost Channa punctatus (Bloch) in response to melatonin under two photothermal conditions. Fish Physiol. Biochem. 5, 187–190. Kukkonen, S., and Laitinen, J. (1987). Melatoniinierityksen vuodenaikaiset muutokset kirjolohella. Suomen Ela¨inla¨a¨ka¨rilehti 93, 359–361. Kulczykowska, E. (1995). Arginine vasotocin—melatonin interactions in fish: A hypothesis. Rev. Fish Biol. Fish 5, 96–102. Ku¨hn, E. R., Corneillie, S., and Ollevier, F. (1986). Circadian variations in plasma osmolality, electrolytes, glucose, and cortisol in carp (Cyprinus carpio). Gen. Comp. Endocrinol. 61, 459–468. Laidley, C. W., and Leatherland, J. F. (1988). Circadian studies of plasma cortisol, thyroid hormone, protein, glucose and ion concentration, liver glycogen concentration and liver and spleen weight in rainbow trout, Salmo gairdneri Richardson. Comp. Biochem. Physiol. A 89, 495–503. Max, M., and Menaker, M. (1992). Regulation of melatonin production by light, darkness, and temperature in the trout pineal. J. Comp. Physiol. A 170, 479–489. Meier, A. H., and Fivizzani, A. J. (1975). Changes in the daily rhythm
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250 of plasma corticosterone concentration related to seasonal conditions in the white-throated sparrow, Zonotrichia albicollis. Proc. Soc. Exp. Biol. Med. 150, 356–362. Nichols, D. J., and Weisbart, M. (1984). Plasma cortisol concentrations in Atlantic Salmon, Salmo salar: Episodic variations, diurnal change, and short term response to adrenocorticotrophic hormone. Gen. Comp. Endocrinol. 56, 169–176. Pang, S.-F., Brown, G. M., Grota, L. J., Chambers, J. W., and Rodman, R. L. (1977). Determination of N-acetylserotonin and melatonin activities in the pineal gland, retina, Harderian gland, brain and serum of rat and chickens. Neuroendocrinology 23, 1–13. Pavlidis, M., Berry, M., Divanach, P., and Kentouri, M. (1997). Diel pattern of haematocrit, serum metabolites, osmotic pressure, electrolytes and thyroid hormones in sea bass and sea bream. Aquacult. Int. 5, 237–247. Peter, R. E., Hontela, A., Cook, A. F., and Paulencu, C. R. (1978). Daily cycles in serum cortisol levels in the goldfish: Effects of photoperiod, temperature, and sexual condition. Can. J. Zool. 56, 2443– 2448. Pickering, A. D., and Pottinger, T. G. (1983). Seasonal and diel changes in plasma cortisol levels of the brown trout, Salmo trutta L. Gen. Comp. Endocrinol. 49, 232–239. Rance, T. A., Baker, B. I., and Werbley, G. (1982). Variations in plasma cortisol concentrations over a 24-hour period in the rainbow trout Salmo gairdneri. Gen. Comp. Endocrinol. 48, 269–274. Randall, C. F., Trush, M., and Bromage, N. (1991). Absence of an endogenous component regulating melatonin secretion in the rainbow trout. In ‘‘Advances in Pineal Research: 5’’ (J. Arendt and P. Pe´vet, Eds.), pp. 279–281. Libbey, London. Randall, C. F., Bromage, N. R., Thorpe, J. E., Miles, M. S., and Muir, J. S. (1995). Melatonin rhythms in Atlantic salmon (Salmo salar) maintained under natural and out-of-phase photoperiods. Gen. Comp. Endocrinol. 98, 73–86. Redgate, E. S. (1974). Neural control of pituitary-adrenal activity in Cyprinus carpio. Gen. Comp. Endocrinol. 22, 35–41.
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Pavlidis et al. Reiter, R. (1991). Pineal melatonin: Cell biology of its synthesis and of its physiological interactions. Endocrine Rev. 11, 151–180. Rollag, M. D., and Niswender, G. D. (1976). Radioimmunoassay of serum concentrations of melatonin in sheep exposed to different lighting regimens. Endocrinology 98, 482–489. Scott, A. P., Mackenzie, D. N., and Stacey, N. E. (1984). Endocrine changes during natural spawning of the white sucker Catostomus commersoni. II. Steroid hormones. Gen. Comp. Endocrinol. 56, 349–359. Spieler, R. E., and Noeske, T. A. (1984). Effects of photoperiod and feeding schedule on diel variations of locomotor activity, cortisol, and thyroxine in goldfish. Trans. Am. Fish. Soc. 115, 528–539. Stokkan, K.-A., Vaughan, M. K., Reiter, R. J., Folkow, L. P., Mårtensson, P.-E., Sager, G., Lydersen, C., and Blix, A. S. (1995). Pineal and thyroid functions in newborn seals. Gen. Comp. Endocrinol. 98, 321–331. Thibault, C., Falcon, J., Greenhouse, S. S., Lowery, C. A., Gern, W. A., and Collin, J. P. (1993). Regulation of melatonin production by pineal photoreceptory cells: role of cyclic nucleotides in trout (Oncorhynhus mykiss). J. Neurochem. 61, 332–339. Valtonen, M., Laitinen, J. T., and Eriksson, L. (1993). Renal melatonin excretion in sheep is enhanced by water diuresis. J. Endocrinol. 138, 445–450. Underwood, H. (1985). Pineal melatonin rhythms in the lizard Anolis carolinensis: Effects of light and temperature cycles. J. Comp. Physiol. A 157, 57–65. Zachmann, A., Knijff, S. C. M., Ali, M. A., and Anctil, M. (1992a). Effects of photoperiod and different intensities of light exposure on melatonin levels in the blood, pineal organ, and retina of the brook trout (Salvelinus fontinalis Mitchill). Can. J. Zool. 70, 25–29. Zachmann, A., Falco´n, J., Knijff, S. C. M., Bolliet, V., and Ali, M. A. (1992b). Effects of photoperiod and temperature on rhythmic melatonin secretion from the pineal organ of the white sucker (Catostomus commersoni) in vitro. Gen. Comp. Endocrinol. 86, 26–33.