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Locomotor, feeding and melatonin daily rhythms in sharpsnout seabream (Diplodus puntazzo)
Physiology & Behavior 88 (2006) 167 – 172
Locomotor, feeding and melatonin daily rhythms in sharpsnout seabream (Diplodus puntazzo) L.M. Vera, J.A. Madrid, F.J. Sánchez-Vázquez ⁎ Department of Physiology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain Received 1 December 2005; received in revised form 22 March 2006; accepted 29 March 2006
Abstract Sharpsnout seabream is a marine teleost of increasing interest for Mediterranean aquaculture, but there is still a lack of information regarding its circadian organization. In this study, we have investigated sharpsnout seabream locomotor activity, feeding and plasma melatonin daily rhythms under a 12:12-h LD cycle, as well as the persistence of locomotor activity circadian rhythmicity under constant light (LL) conditions. When submitted to an LD cycle, most sharpsnout seabream displayed a diurnal locomotor pattern, with an average 74% of activity recorded during daytime. However, along the experiment 40% of fish spontaneously changed their locomotor rhythm phasing and became nocturnal. Feeding behaviour, nevertheless, remained strictly diurnal in all cases, with 97% of food demands being made during the light period. Free-running locomotor rhythms were recorded in one third of the fish kept under LL. Daily plasma melatonin levels displayed a rhythmic profile, with low daytime values (111 pg/ml) and high nighttime concentrations (791 pg/ml). Taken together, these results evidence a high degree of plasticity for sharpsnout seabream activity patterns, as well as phasing independence of locomotor and feeding rhythms. Finally, the existence of a well-defined daily rhythm of plasma melatonin was found. © 2006 Elsevier Inc. All rights reserved. Keywords: Sharpsnout seabream; Locomotor activity; Feeding behavior; Melatonin
1. Introduction The environmental light–dark (LD) cycle is the dominant synchronizer of biological rhythms in vertebrates [1,2]. In fish, daily behavioural patterns are also affected by this cycle, which determines their diurnal or nocturnal way of life [3], though some fish species are endowed with a flexible circadian system that allows them to change their phasing [4,5]. A difference has been established between marine species, presumably having a strong circadian system, and freshwater fishes that would be endowed with a more plastic one [6]. In the latter case, the circadian clock could mainly serve for anticipating daily events such as dusk/ dawn or food availability. Thanks to this particular feature, the daily distribution of locomotor and/or feeding behaviour does not have to be necessarily restricted to the light or dark phase. Hence, different individuals of a given species can be active at different
⁎ Corresponding author. Tel.: +34 968 367004; fax: +34 968 369663. E-mail address: [email protected] (F.J. Sánchez-Vázquez). 0031-9384/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2006.03.031
phases of the LD cycle, and any single animal can switch from a diurnal to a nocturnal activity pattern and back. In order to study the existence of an endogenous control of locomotor and feeding rhythms, animals have to be isolated from external time cues that could synchronize their biological rhythms. To this end, circadian rhythms are monitored while fish are reared in a “chronolaboratory” under constant conditions. In such situation, if an endogenous pacemaker exists, circadian rhythms are expected to persist and free-run. The pineal organ is considered a key component of the vertebrate circadian system [7], and is thought to be involved in the timing and control of rhythmic functions and behaviors, including locomotor and feeding activity [8]. The pineal organ transduces environmental information, mainly the photoperiod, into nervous and hormonal signals like melatonin, which acts as an internal “zeitgeber” in vertebrates [9]. Sharpsnout seabream is a species with great potential for aquaculture, and some studies have already been conducted addressing relevant issues such as parasitism, population structure, genetic differentiation, tissue fatty acid composition, larval growth
L.M. Vera et al. / Physiology & Behavior 88 (2006) 167–172
2.4. Melatonin analysis Melatonin was determined using a commercial radioimmunoassay (RIA) kit (IBL, Hamburg, Germany) [14]. In order to validate the method for sharpsnout seabream samples, we tested the existence of parallelism between plasma serial twofold dilutions and the standard curve. 2.5. Data analysis Locomotor activity records were stored in a computer and analyzed using a software package for chronobiological studies (El Temps®, Prof. A. Díez-Noguera, University of Barcelona). The period length (tau) of free-running rhythms was determined by periodogram analysis at a confidence level of 95%. Data 80
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2.3.1. Experiment 1: locomotor activity and feeding rhythms The walls of all aquaria were covered with black plastic to prevent animals from seeing each other. Ten fish were first reared during 3 weeks under a 12:12-h LD cycle, with lights on at 0800 h and off at 2000 h, and then exposed to LL conditions to investigate the possible existence of endogenous rhythmicity of locomotor activity. During LL, fish were fed at random hours to avoid feeding from acting as a synchronizer. Special care was taken to make sure that external stimuli did not disturb experimental animals.
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To record locomotor activity each aquarium was equipped with an infrared photocell (Omron, E35-AD62, Japan) placed 10 cm away from the bottom and 7 cm away from the lateral wall. Every time a fish interrupted the infrared light beam, it produced an output signal that was recorded and stored by a computer in 10-min bins. Feeding activity was recorded using a commercial feeder (EHEIM, model 3581, Germany) modified in our laboratory to allow for self-feeding [13] and filled with a commercial feed (Europa 22, Trow España S.A., Spain). In order to prevent interference between locomotor and feeding recordings, infrared photocells and self-feeders were placed in opposite sides of the aquaria [4].
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The present study was carried out in a chronobiology laboratory isolated from external conditions, and using a total of 58 sharpsnout seabream (187 ± 37 g body weight). Animals came from the “Centro de Recursos Marinos (San Pedro del Pinatar, Murcia, Spain)” research center. Fish were individually placed in a series of interconnected, 60-l glass aquaria, with water being recirculated among them and filtered through a biological filter and a skimmer. Artificial seawater was prepared by adding synthetic sea salt (Ocean Fish, Prodac, Italy) to well-aerated tap water. Water salinity and temperature (using an electronic thermostat placed in the laboratory) were maintained constant at 3.4% and 22–23 °C, respectively. Each aquarium was fitted with an oxygen diffuser. Lighting was provided by a white fluorescent tube (GRO-LUX, 40 W, Germany), placed over each aquarium about 15 cm above the water surface and delivering 1100 lx. The photoperiod was controlled by a programmable clock.
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2.3.2. Experiment 2: plasma melatonin rhythms Forty eight sharpsnout seabream, housed at the “Centro de Recursos Marinos” research center, were used for this experiment. Animals were kept in an open system with a constant flow of marine water under the natural photoperiod (15L:9D). Water temperature was 24.5 °C. Blood samples were taken every 3 h for melatonin analysis. During the dark phase, this was done under a dim red light and after covering the fish head with aluminium foil. Before blood extraction, animals were individually anaesthetized with water containing 0.5 ml/l of 2-phenoxiethanol. Blood samples were centrifugated, and the resulting plasma samples were frozen and stored at − 80 °C until analysis.
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and food preferences (e.g. 10–12). However, the circadian organization of its biological rhythms has not been yet investigated. The main objective of this study was to examine sharpsnout seabream (Diplodus puntazzo) locomotor activity and feeding daily rhythms under a 12:12-h LD cycle, as well as the persistence of circadian rhythmicity of locomotor activity under constant light (LL) conditions. Furthermore, plasma melatonin rhythms were also investigated in this species.
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Time of day (h) Fig. 1. Average diel profile of sharpsnout seabream locomotor (A) and feeding (B) activity under a 12:12 LD cycle. The white and black bars at the top of each graph indicate the light and dark periods, respectively. Discontinuous lines indicate the beginning and the end of the photophase.
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activity occurring during the photophase. The average diel locomotion profile appeared as a relatively square wave in which activity occurred during the photophase (Fig. 1A). Interestingly, the level of activity started to rise 1 h before lights on and it persisted immediately after lights off, progressively declining back to dark levels during the first hour of the scotophase. In relation to feeding, a few weeks were needed for fish to learn how to use self-feeders and establishing a stable and regular feeding pattern. Under a 12:12-h LD cycle, sharpsnout seabream displayed a strictly diurnal feeding behaviour, with 97% of food demands being made during the photophase. No
analysis was performed with the aid of the statistical program SPSS and Microsoft Excel. Statistical differences between mean melatonin levels were analyzed by one-way analysis of variance (ANOVA) followed by a Duncan's test, with P b 0.05 taken as the statistically significant threshold. 3. Results 3.1. Locomotor activity and feeding rhythms Sharpsnout seabream reared under 12:12-h LD displayed a mostly diurnal locomotor activity pattern, with 74% of their LOCOMOTOR
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Time of day (h) Fig. 2. Locomotor (left) and feeding (right) actograms from representative sharpsnout seabream showing (A) a dual pattern of locomotor activity with diurnal feeding and (B) diurnal patterns of both locomotion and feeding. Actograms are double-plotted for better visualization. The white and black bars at the top of each graph indicate the light and dark periods, respectively.
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3.2. Plasma melatonin rhythms Parallelism of plasma serial dilutions and standard curve was observed in sharpsnout seabream, thus validating the RIA for plasma samples of this species (Fig. 4). Intra-assay coefficient of variation (%V) was 12% and the limit of detection (LD) 3.5 pg/ml. Under a natural 15L:9D photoperiod, sharpsnout seabream plasma melatonin concentrations exhibited rhythmic oscillations, with a night peak and statistically significant differences between day and night levels (ANOVA, P b 0.05). Average daytime melatonin concentration was 146.6 ± 32.1 pg/ml, compared to 697.7 ± 57.4 pg/ml at nighttime. Plasma melatonin values seemed to rise 1 h before the beginning of the scotophase,
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feeding activity at all was detected before lights on, though some scarce feeding events were recorded during the first hour of the scotophase (Fig. 1B). Along the experiment, locomotor activity patterns varied from one individual to another. Some animals (40% of fish used in this experiment) spontaneously switched from diurnal to nocturnal activity, i.e., they showed a dual pattern of locomotor activity. Thus, for example, the fish depicted in Fig. 2A exhibited a mostly diurnal behaviour during the first 7 days (72% of locomotor activity during the photophase), and then changed to a nocturnal pattern, with 70% of its locomotor activity being recorded at night. By contrast, feeding behaviour remained strictly diurnal, which suggests a preference for daytime feeding regardless the locomotor activity pattern. Notwithstanding, most fish did exhibit diurnal activity patterns for both locomotion and feeding (e.g., Fig. 2B). In the absence of any external daily synchronizer (LL), 3 out of 10 sharpsnout seabream exhibited self-sustained circadian rhythms. Locomotor activity free ran with an average tau of 23.9 ± 0.9 h. Two of the fish showing rhythmicity in LL had been diurnal in LD, while the other was nocturnal. This fact suggested that whether animals displayed their activity either during the photophase or scotophase in LD did not seem to affect the existence of rhythmicity in LL. Curiously enough, when the LD cycle was suppressed and fish were exposed to LL conditions, the daily activity of some animals increased, during both their subjective day and night, albeit circadian rhythmicity was maintained (Fig. 3). Sharpsnout seabream average activity increased from 4496 ± 232 counts/day at LD to 8753 ± 623 counts/day after animals were exposed to LL, the difference being statistically significant (t-Student, P = 0.001).
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melatonin (pg/ml) Fig. 4. Validation of the radioimmunoassay method for sharpsnout seabream plasma samples. The parallelism between the standard curve (black diamonds) and plasma serial dilutions (white circles) demonstrates the validity of the assay for this species.
L.M. Vera et al. / Physiology & Behavior 88 (2006) 167–172
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Time of day (h) Fig. 5. Daily plasma melatonin rhythm in sharpsnout seabream under a 15L:9D cycle. Values represent the mean ± S.E.M. of six animals. Different letters indicate statistically significant differences between sampling points (ANOVA, Duncan's test, P b 0.05).
presumably in anticipation of the approaching day, although this elevation was not statistically significant (Fig. 5). 4. Discussion This study provides the first results concerning locomotor, feeding and melatonin rhythms in sharpsnout seabream (D. puntazzo), a marine species with great prospects for aquaculture. Sharpsnout seabream submitted to an LD cycle display a mainly diurnal behaviour, although some individuals may change to a nocturnal locomotor activity pattern. Feeding patterns, however, remained strictly diurnal in all cases investigated. In addition, free-running locomotor rhythms were observed in some fish under LL, indicating the existence of endogenous timing mechanisms controlling locomotor rhythms. The spontaneous change from diurnal to nocturnal locomotor activity of some sharpsnout seabream supports the widely accepted theory that fish have a flexible circadian system [6]. In most vertebrates, locomotion and feeding are strictly restricted to either the light or dark phase, but fish include many of the exceptions to this general rule. For instance, in a study analyzing the circadian rhythms of goldfish (Carassius auratus) locomotor activity under laboratory conditions, 83% of the animals behaved as diurnal, 7% were nocturnal and 10% showed constant levels of activity throughout the entire day [15]. Lake chubs (Couesius plumbeus) behave as diurnal when reared in the laboratory [16], but they exhibit nocturnal feeding habits under natural conditions [17]. Steelhead trout (Oncorhynchus mykiss) also shows interindividual locomotor activity differences under the same experimental conditions [18], and a relatively independent phasing between feeding and locomotion rhythms depending on the water depth [19]. As to marine species, European sea bass (Dicentrarchus labrax) feeding from self-feeders would demand food during the light or dark period under the same laboratory conditions [20]. In our experiments, although locomotor activity in some fish shifted to nocturnal, feeding remained diurnal. A similar dissociation between both types of activity was observed by Sánchez-Vázquez et al. in goldfish [4]. These authors advanced a tentative explanation to the effect that since self-feeders give animals free access to food, freeing them from having to engage
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in an active search, they may or may not actively swim around the aquarium while feeding. Circadian rhythms under continuous light or dark have been demonstrated in several fish species [19,21–25], although the circadian activity pattern seems to be labile and shows interindividual variations. Some fish may lose free-running rhythms shortly after constant conditions are established [26– 28], while others may be completely arrhythmic. It has been proposed that there are two coupled oscillators synchronized to dawn and dusk [29]. When fish are reared under constant conditions, both oscillators are uncoupled and locomotor rhythms free run with a given period, which could cause arrhythmicity in some cases. In sharpsnout sea bream, free-running locomotor rhythms under constant conditions point to the existence of an endogenous control of these rhythms. In our experiments, however, only one-third of the animals displayed a self-sustained locomotor rhythm under LL, a value that is consistent with those obtained for other fish species. For instance, only 41.6% of tench showed a significant tau value under DD [21], whereas only 16.7% of trout exhibited circadian rhythmicity under dim LL [19]. It should be noted that in our experiments no selection of fish whatsoever was made and meals were provided at random times in order to prevent locomotor rhythms from being synchronized by periodic food access. Plasma melatonin levels in sharpsnout seabream show the same daily profile as seen in most vertebrates: in all species investigated, melatonin production by the pineal organ is lowest during daytime and peaks at night [30]. Melatonin synthesis by the pineal is directly related to plasma melatonin levels [31]. In sharpsnout sea bream, plasma melatonin concentrations at both light (111.1 ± 30.6 pg/ml at mid-light, ML) and dark (791.3 ± 114.7 pg/ml at mid-darkness, MD) phases were higher than those described for other fish species such as European sea bass (D. labrax) [32] and tench (Tinca tinca) [14], with maximum reported levels of 255 ± 40 pg/ml and 255 ± 66 pg/ml, respectively. Maximum MD titters of around 80 pg/ml [33] have been reported for sole (Solea senegalensis), whereas MD plasma melatonin concentrations as low as 28± 2 pg/ml [34] were found in turbot (Scophthalmus maximus). However, some authors have found higher values in other fish species, such as the common carp (Cyprinus carpio), with plasma melatonin concentrations of 220– 540 pg/ml for the dark phase and 23–104 pg/ml for the light phase [35], and rainbow trout (O. mykiss), with values close to 800 pg/ ml in summer [36]. In principle, there could be several reasons for this variability; possible intrinsic interspecies variation and a number of environmental factors (i.e. photoperiod length, temperature and light intensity). While the duration of nocturnal melatonin synthesis is determined by the photoperiod, the amplitude is thought to be influenced by light intensity [14,37,38]. Indeed, temperature has been reported to affect the amplitude of the nocturnal melatonin peak; melatonin synthesis increases with increasing temperature up to an optimum temperature beyond which it declines [39,40]. In this sense, the fact that sharpsnout seabream plasma samples for these experiments were taken around the summer solstice in the South East of Spain (with long photoperiod and high daytime temperature and light intensity) most surely affected melatonin levels.
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In conclusion, this study presents the first results on sharpsnout seabream locomotor activity, feeding and melatonin daily rhythms. Most animals displayed a diurnal, albeit quite plastic, locomotor activity pattern, as some fish spontaneously shifted from a diurnal to a nocturnal pattern. By contrast, irrespective of whether or not locomotor activity had switched to nocturnal, feeding activity remained strictly diurnal, pointing to a phase independence between locomotor and feeding activity. Furthermore, daily plasma melatonin rhythms suggest a role for this hormone in transducing daily environmental cycles (i.e., light and temperature), acting as an internal synchronizer for the circadian system of this species. References [1] Challet E, Pevet P. Interactions between photic and nonphotic stimuli to synchronize the master circadian clock in mammals. Front Biosci 2003;8: 246–57. [2] Okimoto DK, Stetson MH. Presence of an intrapineal circadian oscillator in the teleostean family Poeciliidae. Gen Comp Endocrinol 1999;114 (2):304–12. [3] Eriksson L-O. Nocturnalism versus diurnalism—dualism within fish individuals. In: Thorpe JE, editor. Rhythmic Activity of Fishes. New York: Academic Press; 1978. p. 69–89. [4] Sánchez-Vázquez FJ, Madrid JA, Zamora S, Iigo M, Tabata M. Demand feeding and locomotor circadian rhythms in the goldfish, Carassius auratus: dual and independent phasing. Physiol Behav 1996;60(2):665–74. [5] Ali MA, editor. Rhythms in Fishes. New York: Plenum Press; 1992. [6] Reebs SG. Plasticity of diel and circadian activity rhythms in fishes. Rev Fish Biol Fish 2002;12:349–71. [7] Zachmann A, Ali MA, Falcón J. Melatonin and its effects in fishes: an overview. In: Ali MA, editor. Rhythms in Fishes. New York: Plenum Press; 1992. [8] Ekström P, Meissl H. The pineal organ of teleost fishes. Rev Fish Biol Fish 1997;7:199–284. [9] Falcón J. Cellular circadian clocks in the pineal. Prog Neurobiol 1999;58: 121–62. [10] Rueda FM, Hernández MD, Egea MA, Aguado F, García B, Martínez FJ. Differences in tissue fatty acid composition between reared and wild sharpsnout sea bream, Diplodus puntazzo (Cetti, 1777). Br J Nutr 2001;86: 617–22. [11] Loy A, Bertelletti M, Costa C, Ferlin L, Cataudella S. Shape changes and growth trajectories in the early stages of three species of the genus Diplodus (Perciformes, Sparidae). J Morphol 2001;250(1):24–33. [12] Torrejón Atienza M, Chatzifotis S, Divanach P. Macronutrient selection by sharp snout seabream (Diplodus puntazzo). Aquaculture 2004;232 (1–4):481–91. [13] Herrero MJ, Pascual M, Madrid JA, Sánchez-Vázquez FJ. Demandfeeding rhythms and feeding-entrainment of locomotor activity rhythms in tench (Tinca tinca). Physiol Behav 2005;84:595–605. [14] Vera LM, López-Olmeda JF, Bayarri MJ, Madrid JA, Sánchez-Vázquez FJ. Influence of Light intensity on plasma melatonin and locomotor activity rhythms in. Tench Chronobiol Int 2005;22(1):67–78. [15] Iigo M, Tabata M. Circadian rhythms of locomotor activity in the goldfish Carassius auratus. Physiol Behav 1996;60:775–81. [16] Kavaliers M, Ross DM. Twilight and day length affects the seasonality of entrainment and endogenous circadian rhythms in a fish, Couesius plumbeus. Can J Zool 1981;59:1326–34. [17] Emery AR. Preliminary comparisons of day and night habits of freshwater fish in Notario lakes. J Fish Res Board Can 1973;30:761–74. [18] Alanärä A, Bränäs E. Diurnal and nocturnal feeding activity in Arctic char (Salvelinus alpinus) and rainbow trout (Oncorhynchus mykiss). Can J Fish Aqua Sci 1997;54:2894–900.
[19] Sánchez-Vázquez FJ, Tabata M. Circadian rhythms of demand-feeding and locomotor activity in rainbow trout. J Fish Biol 1998;52:255–67. [20] Sánchez-Vázquez FJ, Madrid JA, Zamora S. Circadian rhythms of feeding activity in sea bass Dicentrarchus labrax L.: dual phasing capacity of diel demand-feeding pattern. J Biol Rhythms 1995;10:256–66. [21] Herrero MJ, Madrid JA, Sánchez-Vázquez FJ. Entrainment to light of circadian activity rhythms in tench (Tinca tinca). Chronobiol Int 2003;20 (6):1001–17. [22] Lissmann HW, Schwassmann HO. Activity rhythm of an electric fish, Gymnorhamphichthys hypostomus. Z Vergl Physiol 1965;51:153–70. [23] Kavaliers M. Pineal involvement in the control of circadian rhythmicity in the lake chub, Couesius plumbeus. J Exp Zool 1979;209:33–40. [24] Nishi G. Locomotor activity rhythm in two wrasses, Halichoeres tenuispinnis and Pteragogus flegellifera, under various light conditions. Japan. J Ichthyol 1989;36:350–6. [25] Hurd MW, Debruyne J, Straume M, Cahill GM. Circadian rhythms of locomotor activity in zebrafish. Physiol Behav 1998;65(3):465–72. [26] Davis RE. Daily ‘predawn’ peak of locomotion in bluegill and largemouth bass. Anim Behav 1963;12:272–83. [27] Godin J-GJ. Circadian rhythm of swimming activity in juvenile pink salmon (Oncorhynchus gorbuscha). Mar Biol 1981;64:341–9. [28] Nishi G. Locomotor activity rhythm in four wrasse species under varying light conditions. Jpn J Ichtyol 1990;37:170–81. [29] Andreasson S. Seasonal changes in diel activity of Cottus poecilopus Heckel and C. gobio (Pisces) at the artic circle. Oikos 1973;24:16–23. [30] Collin JP, Voisin P, Falcón J, Faure JP, Brisson P, Defaye JR. Pineal transducers in the course of evolution: molecular organization, rhythmic metabolic activity and role. Arch Histol Cytol 1989;52:441. [31] Gern WA, Owens DW, Ralph CL. Plasma melatonin in the trout: day– night change demonstrated by radioimmunoassay. Gen Comp Endocrinol 1978;34:453–8. [32] Sánchez-Vázquez FJ, Iigo M, Madrid JA, Zamora S, Tabata M. Daily cycles in plasma and ocular melatonin in demand-fed sea bass, Dicentrarchus labrax L. Comp Physiol B 1997;167:409–15. [33] Bayarri MJ, Muñoz-Cueto JA, López-Olmeda JF, Vera LM, Rol de Lama MA, Madrid JA, Sánchez-Vázquez FJ. Daily locomotor activity and melatonin rhythms in Senegal sole (Solea senegalensis). Physiol Behav 2004;81:577–83. [34] Rebollar PG, Ubilla E, Peleteiro JB, Agapito MT, Alvariño JMR. Determination of plasma melatonin levels by enzyme-linked immunosorbent assay (EIA) in turbot (Scophtalmus maximus L.) and tench (Tinca tinca L.). J Physiol Biochem 1999;55(4):341–8. [35] Kezuka H, Furukawa K, Aida K, Hanyu I. Daily cycles in plasma melatonin levels under long or short photoperiod in the common carp, Cyprinus carpio. Gen Comp Endocrinol 1988;72(2):296–302. [36] Masuda T, Iigo M, Mizusawa K, Naruse K, Oishi T, Aida K, et al. Variations in plasma melatonin levels of the rainbow trout (Oncorhynchus mykiss) under various light and temperature conditions. Zoolog Sci 2003;20: 1011–6. [37] Gern WA, Greenhouse SS. Examination of in vitro melatonin secretion from superfused trout (Salmo gairdneri) pineal organs maintained under diel illumination or continuous darkness. Gen Comp Endocrinol 1988;71: 163–74. [38] Meyer WE, Millam JR. Plasma melatonin levels in Japanese quail exposed to dim light are determined by subjective interpretation of day and night, not light intensity. Gen Comp Endocrinol 1991;82:377–85. [39] Falcón J, Collin JP. Photoreceptors in the pineal of lower vertebrates: functional aspects. Experientia 1989;45:909–13. [40] Max M, Menaker M. Regulation of melatonin production by light, darkness, and temperature in the trout pineal. J Comp Physiol 1992;170A: 479–89.
Locomotor, feeding and melatonin daily rhythms in sharpsnout seabream (Diplodus puntazzo) L.M. Vera, J.A. Madrid, F.J. Sánchez-Vázquez ⁎ Department of Physiology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain Received 1 December 2005; received in revised form 22 March 2006; accepted 29 March 2006
Abstract Sharpsnout seabream is a marine teleost of increasing interest for Mediterranean aquaculture, but there is still a lack of information regarding its circadian organization. In this study, we have investigated sharpsnout seabream locomotor activity, feeding and plasma melatonin daily rhythms under a 12:12-h LD cycle, as well as the persistence of locomotor activity circadian rhythmicity under constant light (LL) conditions. When submitted to an LD cycle, most sharpsnout seabream displayed a diurnal locomotor pattern, with an average 74% of activity recorded during daytime. However, along the experiment 40% of fish spontaneously changed their locomotor rhythm phasing and became nocturnal. Feeding behaviour, nevertheless, remained strictly diurnal in all cases, with 97% of food demands being made during the light period. Free-running locomotor rhythms were recorded in one third of the fish kept under LL. Daily plasma melatonin levels displayed a rhythmic profile, with low daytime values (111 pg/ml) and high nighttime concentrations (791 pg/ml). Taken together, these results evidence a high degree of plasticity for sharpsnout seabream activity patterns, as well as phasing independence of locomotor and feeding rhythms. Finally, the existence of a well-defined daily rhythm of plasma melatonin was found. © 2006 Elsevier Inc. All rights reserved. Keywords: Sharpsnout seabream; Locomotor activity; Feeding behavior; Melatonin
1. Introduction The environmental light–dark (LD) cycle is the dominant synchronizer of biological rhythms in vertebrates [1,2]. In fish, daily behavioural patterns are also affected by this cycle, which determines their diurnal or nocturnal way of life [3], though some fish species are endowed with a flexible circadian system that allows them to change their phasing [4,5]. A difference has been established between marine species, presumably having a strong circadian system, and freshwater fishes that would be endowed with a more plastic one [6]. In the latter case, the circadian clock could mainly serve for anticipating daily events such as dusk/ dawn or food availability. Thanks to this particular feature, the daily distribution of locomotor and/or feeding behaviour does not have to be necessarily restricted to the light or dark phase. Hence, different individuals of a given species can be active at different
⁎ Corresponding author. Tel.: +34 968 367004; fax: +34 968 369663. E-mail address: [email protected] (F.J. Sánchez-Vázquez). 0031-9384/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2006.03.031
phases of the LD cycle, and any single animal can switch from a diurnal to a nocturnal activity pattern and back. In order to study the existence of an endogenous control of locomotor and feeding rhythms, animals have to be isolated from external time cues that could synchronize their biological rhythms. To this end, circadian rhythms are monitored while fish are reared in a “chronolaboratory” under constant conditions. In such situation, if an endogenous pacemaker exists, circadian rhythms are expected to persist and free-run. The pineal organ is considered a key component of the vertebrate circadian system [7], and is thought to be involved in the timing and control of rhythmic functions and behaviors, including locomotor and feeding activity [8]. The pineal organ transduces environmental information, mainly the photoperiod, into nervous and hormonal signals like melatonin, which acts as an internal “zeitgeber” in vertebrates [9]. Sharpsnout seabream is a species with great potential for aquaculture, and some studies have already been conducted addressing relevant issues such as parasitism, population structure, genetic differentiation, tissue fatty acid composition, larval growth
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2.4. Melatonin analysis Melatonin was determined using a commercial radioimmunoassay (RIA) kit (IBL, Hamburg, Germany) [14]. In order to validate the method for sharpsnout seabream samples, we tested the existence of parallelism between plasma serial twofold dilutions and the standard curve. 2.5. Data analysis Locomotor activity records were stored in a computer and analyzed using a software package for chronobiological studies (El Temps®, Prof. A. Díez-Noguera, University of Barcelona). The period length (tau) of free-running rhythms was determined by periodogram analysis at a confidence level of 95%. Data 80
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2.3.1. Experiment 1: locomotor activity and feeding rhythms The walls of all aquaria were covered with black plastic to prevent animals from seeing each other. Ten fish were first reared during 3 weeks under a 12:12-h LD cycle, with lights on at 0800 h and off at 2000 h, and then exposed to LL conditions to investigate the possible existence of endogenous rhythmicity of locomotor activity. During LL, fish were fed at random hours to avoid feeding from acting as a synchronizer. Special care was taken to make sure that external stimuli did not disturb experimental animals.
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To record locomotor activity each aquarium was equipped with an infrared photocell (Omron, E35-AD62, Japan) placed 10 cm away from the bottom and 7 cm away from the lateral wall. Every time a fish interrupted the infrared light beam, it produced an output signal that was recorded and stored by a computer in 10-min bins. Feeding activity was recorded using a commercial feeder (EHEIM, model 3581, Germany) modified in our laboratory to allow for self-feeding [13] and filled with a commercial feed (Europa 22, Trow España S.A., Spain). In order to prevent interference between locomotor and feeding recordings, infrared photocells and self-feeders were placed in opposite sides of the aquaria [4].
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The present study was carried out in a chronobiology laboratory isolated from external conditions, and using a total of 58 sharpsnout seabream (187 ± 37 g body weight). Animals came from the “Centro de Recursos Marinos (San Pedro del Pinatar, Murcia, Spain)” research center. Fish were individually placed in a series of interconnected, 60-l glass aquaria, with water being recirculated among them and filtered through a biological filter and a skimmer. Artificial seawater was prepared by adding synthetic sea salt (Ocean Fish, Prodac, Italy) to well-aerated tap water. Water salinity and temperature (using an electronic thermostat placed in the laboratory) were maintained constant at 3.4% and 22–23 °C, respectively. Each aquarium was fitted with an oxygen diffuser. Lighting was provided by a white fluorescent tube (GRO-LUX, 40 W, Germany), placed over each aquarium about 15 cm above the water surface and delivering 1100 lx. The photoperiod was controlled by a programmable clock.
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2.3.2. Experiment 2: plasma melatonin rhythms Forty eight sharpsnout seabream, housed at the “Centro de Recursos Marinos” research center, were used for this experiment. Animals were kept in an open system with a constant flow of marine water under the natural photoperiod (15L:9D). Water temperature was 24.5 °C. Blood samples were taken every 3 h for melatonin analysis. During the dark phase, this was done under a dim red light and after covering the fish head with aluminium foil. Before blood extraction, animals were individually anaesthetized with water containing 0.5 ml/l of 2-phenoxiethanol. Blood samples were centrifugated, and the resulting plasma samples were frozen and stored at − 80 °C until analysis.
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and food preferences (e.g. 10–12). However, the circadian organization of its biological rhythms has not been yet investigated. The main objective of this study was to examine sharpsnout seabream (Diplodus puntazzo) locomotor activity and feeding daily rhythms under a 12:12-h LD cycle, as well as the persistence of circadian rhythmicity of locomotor activity under constant light (LL) conditions. Furthermore, plasma melatonin rhythms were also investigated in this species.
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Time of day (h) Fig. 1. Average diel profile of sharpsnout seabream locomotor (A) and feeding (B) activity under a 12:12 LD cycle. The white and black bars at the top of each graph indicate the light and dark periods, respectively. Discontinuous lines indicate the beginning and the end of the photophase.
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activity occurring during the photophase. The average diel locomotion profile appeared as a relatively square wave in which activity occurred during the photophase (Fig. 1A). Interestingly, the level of activity started to rise 1 h before lights on and it persisted immediately after lights off, progressively declining back to dark levels during the first hour of the scotophase. In relation to feeding, a few weeks were needed for fish to learn how to use self-feeders and establishing a stable and regular feeding pattern. Under a 12:12-h LD cycle, sharpsnout seabream displayed a strictly diurnal feeding behaviour, with 97% of food demands being made during the photophase. No
analysis was performed with the aid of the statistical program SPSS and Microsoft Excel. Statistical differences between mean melatonin levels were analyzed by one-way analysis of variance (ANOVA) followed by a Duncan's test, with P b 0.05 taken as the statistically significant threshold. 3. Results 3.1. Locomotor activity and feeding rhythms Sharpsnout seabream reared under 12:12-h LD displayed a mostly diurnal locomotor activity pattern, with 74% of their LOCOMOTOR
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3.2. Plasma melatonin rhythms Parallelism of plasma serial dilutions and standard curve was observed in sharpsnout seabream, thus validating the RIA for plasma samples of this species (Fig. 4). Intra-assay coefficient of variation (%V) was 12% and the limit of detection (LD) 3.5 pg/ml. Under a natural 15L:9D photoperiod, sharpsnout seabream plasma melatonin concentrations exhibited rhythmic oscillations, with a night peak and statistically significant differences between day and night levels (ANOVA, P b 0.05). Average daytime melatonin concentration was 146.6 ± 32.1 pg/ml, compared to 697.7 ± 57.4 pg/ml at nighttime. Plasma melatonin values seemed to rise 1 h before the beginning of the scotophase,
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feeding activity at all was detected before lights on, though some scarce feeding events were recorded during the first hour of the scotophase (Fig. 1B). Along the experiment, locomotor activity patterns varied from one individual to another. Some animals (40% of fish used in this experiment) spontaneously switched from diurnal to nocturnal activity, i.e., they showed a dual pattern of locomotor activity. Thus, for example, the fish depicted in Fig. 2A exhibited a mostly diurnal behaviour during the first 7 days (72% of locomotor activity during the photophase), and then changed to a nocturnal pattern, with 70% of its locomotor activity being recorded at night. By contrast, feeding behaviour remained strictly diurnal, which suggests a preference for daytime feeding regardless the locomotor activity pattern. Notwithstanding, most fish did exhibit diurnal activity patterns for both locomotion and feeding (e.g., Fig. 2B). In the absence of any external daily synchronizer (LL), 3 out of 10 sharpsnout seabream exhibited self-sustained circadian rhythms. Locomotor activity free ran with an average tau of 23.9 ± 0.9 h. Two of the fish showing rhythmicity in LL had been diurnal in LD, while the other was nocturnal. This fact suggested that whether animals displayed their activity either during the photophase or scotophase in LD did not seem to affect the existence of rhythmicity in LL. Curiously enough, when the LD cycle was suppressed and fish were exposed to LL conditions, the daily activity of some animals increased, during both their subjective day and night, albeit circadian rhythmicity was maintained (Fig. 3). Sharpsnout seabream average activity increased from 4496 ± 232 counts/day at LD to 8753 ± 623 counts/day after animals were exposed to LL, the difference being statistically significant (t-Student, P = 0.001).
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presumably in anticipation of the approaching day, although this elevation was not statistically significant (Fig. 5). 4. Discussion This study provides the first results concerning locomotor, feeding and melatonin rhythms in sharpsnout seabream (D. puntazzo), a marine species with great prospects for aquaculture. Sharpsnout seabream submitted to an LD cycle display a mainly diurnal behaviour, although some individuals may change to a nocturnal locomotor activity pattern. Feeding patterns, however, remained strictly diurnal in all cases investigated. In addition, free-running locomotor rhythms were observed in some fish under LL, indicating the existence of endogenous timing mechanisms controlling locomotor rhythms. The spontaneous change from diurnal to nocturnal locomotor activity of some sharpsnout seabream supports the widely accepted theory that fish have a flexible circadian system [6]. In most vertebrates, locomotion and feeding are strictly restricted to either the light or dark phase, but fish include many of the exceptions to this general rule. For instance, in a study analyzing the circadian rhythms of goldfish (Carassius auratus) locomotor activity under laboratory conditions, 83% of the animals behaved as diurnal, 7% were nocturnal and 10% showed constant levels of activity throughout the entire day [15]. Lake chubs (Couesius plumbeus) behave as diurnal when reared in the laboratory [16], but they exhibit nocturnal feeding habits under natural conditions [17]. Steelhead trout (Oncorhynchus mykiss) also shows interindividual locomotor activity differences under the same experimental conditions [18], and a relatively independent phasing between feeding and locomotion rhythms depending on the water depth [19]. As to marine species, European sea bass (Dicentrarchus labrax) feeding from self-feeders would demand food during the light or dark period under the same laboratory conditions [20]. In our experiments, although locomotor activity in some fish shifted to nocturnal, feeding remained diurnal. A similar dissociation between both types of activity was observed by Sánchez-Vázquez et al. in goldfish [4]. These authors advanced a tentative explanation to the effect that since self-feeders give animals free access to food, freeing them from having to engage
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in an active search, they may or may not actively swim around the aquarium while feeding. Circadian rhythms under continuous light or dark have been demonstrated in several fish species [19,21–25], although the circadian activity pattern seems to be labile and shows interindividual variations. Some fish may lose free-running rhythms shortly after constant conditions are established [26– 28], while others may be completely arrhythmic. It has been proposed that there are two coupled oscillators synchronized to dawn and dusk [29]. When fish are reared under constant conditions, both oscillators are uncoupled and locomotor rhythms free run with a given period, which could cause arrhythmicity in some cases. In sharpsnout sea bream, free-running locomotor rhythms under constant conditions point to the existence of an endogenous control of these rhythms. In our experiments, however, only one-third of the animals displayed a self-sustained locomotor rhythm under LL, a value that is consistent with those obtained for other fish species. For instance, only 41.6% of tench showed a significant tau value under DD [21], whereas only 16.7% of trout exhibited circadian rhythmicity under dim LL [19]. It should be noted that in our experiments no selection of fish whatsoever was made and meals were provided at random times in order to prevent locomotor rhythms from being synchronized by periodic food access. Plasma melatonin levels in sharpsnout seabream show the same daily profile as seen in most vertebrates: in all species investigated, melatonin production by the pineal organ is lowest during daytime and peaks at night [30]. Melatonin synthesis by the pineal is directly related to plasma melatonin levels [31]. In sharpsnout sea bream, plasma melatonin concentrations at both light (111.1 ± 30.6 pg/ml at mid-light, ML) and dark (791.3 ± 114.7 pg/ml at mid-darkness, MD) phases were higher than those described for other fish species such as European sea bass (D. labrax) [32] and tench (Tinca tinca) [14], with maximum reported levels of 255 ± 40 pg/ml and 255 ± 66 pg/ml, respectively. Maximum MD titters of around 80 pg/ml [33] have been reported for sole (Solea senegalensis), whereas MD plasma melatonin concentrations as low as 28± 2 pg/ml [34] were found in turbot (Scophthalmus maximus). However, some authors have found higher values in other fish species, such as the common carp (Cyprinus carpio), with plasma melatonin concentrations of 220– 540 pg/ml for the dark phase and 23–104 pg/ml for the light phase [35], and rainbow trout (O. mykiss), with values close to 800 pg/ ml in summer [36]. In principle, there could be several reasons for this variability; possible intrinsic interspecies variation and a number of environmental factors (i.e. photoperiod length, temperature and light intensity). While the duration of nocturnal melatonin synthesis is determined by the photoperiod, the amplitude is thought to be influenced by light intensity [14,37,38]. Indeed, temperature has been reported to affect the amplitude of the nocturnal melatonin peak; melatonin synthesis increases with increasing temperature up to an optimum temperature beyond which it declines [39,40]. In this sense, the fact that sharpsnout seabream plasma samples for these experiments were taken around the summer solstice in the South East of Spain (with long photoperiod and high daytime temperature and light intensity) most surely affected melatonin levels.
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In conclusion, this study presents the first results on sharpsnout seabream locomotor activity, feeding and melatonin daily rhythms. Most animals displayed a diurnal, albeit quite plastic, locomotor activity pattern, as some fish spontaneously shifted from a diurnal to a nocturnal pattern. By contrast, irrespective of whether or not locomotor activity had switched to nocturnal, feeding activity remained strictly diurnal, pointing to a phase independence between locomotor and feeding activity. Furthermore, daily plasma melatonin rhythms suggest a role for this hormone in transducing daily environmental cycles (i.e., light and temperature), acting as an internal synchronizer for the circadian system of this species. References [1] Challet E, Pevet P. Interactions between photic and nonphotic stimuli to synchronize the master circadian clock in mammals. Front Biosci 2003;8: 246–57. [2] Okimoto DK, Stetson MH. Presence of an intrapineal circadian oscillator in the teleostean family Poeciliidae. Gen Comp Endocrinol 1999;114 (2):304–12. [3] Eriksson L-O. Nocturnalism versus diurnalism—dualism within fish individuals. In: Thorpe JE, editor. Rhythmic Activity of Fishes. New York: Academic Press; 1978. p. 69–89. [4] Sánchez-Vázquez FJ, Madrid JA, Zamora S, Iigo M, Tabata M. Demand feeding and locomotor circadian rhythms in the goldfish, Carassius auratus: dual and independent phasing. Physiol Behav 1996;60(2):665–74. [5] Ali MA, editor. Rhythms in Fishes. New York: Plenum Press; 1992. [6] Reebs SG. Plasticity of diel and circadian activity rhythms in fishes. Rev Fish Biol Fish 2002;12:349–71. [7] Zachmann A, Ali MA, Falcón J. Melatonin and its effects in fishes: an overview. In: Ali MA, editor. Rhythms in Fishes. New York: Plenum Press; 1992. [8] Ekström P, Meissl H. The pineal organ of teleost fishes. Rev Fish Biol Fish 1997;7:199–284. [9] Falcón J. Cellular circadian clocks in the pineal. Prog Neurobiol 1999;58: 121–62. [10] Rueda FM, Hernández MD, Egea MA, Aguado F, García B, Martínez FJ. Differences in tissue fatty acid composition between reared and wild sharpsnout sea bream, Diplodus puntazzo (Cetti, 1777). Br J Nutr 2001;86: 617–22. [11] Loy A, Bertelletti M, Costa C, Ferlin L, Cataudella S. Shape changes and growth trajectories in the early stages of three species of the genus Diplodus (Perciformes, Sparidae). J Morphol 2001;250(1):24–33. [12] Torrejón Atienza M, Chatzifotis S, Divanach P. Macronutrient selection by sharp snout seabream (Diplodus puntazzo). Aquaculture 2004;232 (1–4):481–91. [13] Herrero MJ, Pascual M, Madrid JA, Sánchez-Vázquez FJ. Demandfeeding rhythms and feeding-entrainment of locomotor activity rhythms in tench (Tinca tinca). Physiol Behav 2005;84:595–605. [14] Vera LM, López-Olmeda JF, Bayarri MJ, Madrid JA, Sánchez-Vázquez FJ. Influence of Light intensity on plasma melatonin and locomotor activity rhythms in. Tench Chronobiol Int 2005;22(1):67–78. [15] Iigo M, Tabata M. Circadian rhythms of locomotor activity in the goldfish Carassius auratus. Physiol Behav 1996;60:775–81. [16] Kavaliers M, Ross DM. Twilight and day length affects the seasonality of entrainment and endogenous circadian rhythms in a fish, Couesius plumbeus. Can J Zool 1981;59:1326–34. [17] Emery AR. Preliminary comparisons of day and night habits of freshwater fish in Notario lakes. J Fish Res Board Can 1973;30:761–74. [18] Alanärä A, Bränäs E. Diurnal and nocturnal feeding activity in Arctic char (Salvelinus alpinus) and rainbow trout (Oncorhynchus mykiss). Can J Fish Aqua Sci 1997;54:2894–900.
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