Light and Temperature Cycles as Zeitgebers of Zebrafish (Danio rerio) Circadian Activity Rhythms

Light and temperature cycles are the most important synchronizers of biological rhythms in nature. However, the relative importance of each, especially when they are not in phase, has been poorly studied. The aim of this study was to analyze the entrainment of daily locomotor activity to light and/or temperature cycles in zebrafish. Under two constant temperatures (20°C and 26°C) and 12:12 light-dark (LD) cycles, zebrafish were most active during the day (light) time and showed higher total activity at the warmer temperature, while diurnalism was higher at 20°C than at 26°C (87% and 77%, respectively). Under thermocycles (12:12 LD, 26:20°C thermophase:chryophase or TC), zebrafish daily activity synchronized to the light phase, both when the thermophase and light phase were in phase (LD/TC) or in antiphase (LD/CT). Under constant dim light (3 lux), nearly all zebrafish synchronized to thermocycles (τ=24 h), although activity rhythms (60% to 67% of activity occurred during the thermophase) were not as marked as those observed under the LD cycle. Under constant dim light of 3 lux and constant temperature (22.5°C), 4 of 6 groups of zebrafish previously entrained to thermocycles displayed free‐running rhythms (τ=22.9 to 23.6 h). These results indicate that temperature cycles alone can also entrain zebrafish locomotor activity.     

Does lighting manipulation during incubation affect hatching rhythms and early development of sole?

Light plays a key role in the development of biological rhythms in fish. Previous research on Senegal sole has revealed that both spawning rhythms and larval development are strongly influenced by lighting conditions. However, hatching rhythms and the effect of light during incubation are as yet unexplored. Therefore, the aim of this study was to investigate the impact of the light spectrum and photoperiod on Solea senegalensis eggs and larvae until day 7 post hatching (dph). To this end, eggs were collected immediately after spawning during the night and exposed to continuous light (LL), continuous darkness (DD), or light-dark (LD) 12L:12D cycles of white light (LD(W)), blue light (LD(B); λ(peak)?=?463?nm), or red light (LD(R); λ(peak)?=?685?nm). Eggs exposed to LD(B) had the highest hatching rate (94.5%?±?1.9%), whereas LD(R) and DD showed the lowest hatching rate (54.4%?±?3.9% and 48.4%?±?4.2%, respectively). Under LD conditions, the hatching rhythm peaked by the end of the dark phase, but was advanced in LD(B) (zeitgeber time 8 [ZT8]; ZT0 representing the onset of darkness) in relation to LD(W) and LD(R) (ZT11). Under DD conditions, the same rhythm persisted, although with lower amplitude, whereas under LL the hatching rhythm split into two peaks (ZT8 and ZT13). From dph 4 onwards, larvae under LD(B) showed the best growth and quickest development (advanced eye pigmentation, mouth opening, and pectoral fins), whereas larvae under LD(R) and DD had the poorest performance. These results reveal that developmental rhythms at the egg stage are tightly controlled by light characteristics, underlining the importance of reproducing their natural underwater photoenvironment (LD cycles of blue wavelengths) during incubation and early larvae development of fish.     

Does Lighting Manipulation During Incubation Affect Hatching Rhythms and Early Development of Sole?

Light plays a key role in the development of biological rhythms in fish. Previous research on Senegal sole has revealed that both spawning rhythms and larval development are strongly influenced by lighting conditions. However, hatching rhythms and the effect of light during incubation are as yet unexplored. Therefore, the aim of this study was to investigate the impact of the light spectrum and photoperiod on Solea senegalensis eggs and larvae until day 7 post hatching (dph). To this end, eggs were collected immediately after spawning during the night and exposed to continuous light (LL), continuous darkness (DD), or light-dark (LD) 12L:12D cycles of white light (LD_W), blue light (LD_B; λ_peak?=?463?nm), or red light (LD_R; λ_peak?=?685?nm). Eggs exposed to LD_B had the highest hatching rate (94.5%?±?1.9%), whereas LD_R and DD showed the lowest hatching rate (54.4%?±?3.9% and 48.4%?±?4.2%, respectively). Under LD conditions, the hatching rhythm peaked by the end of the dark phase, but was advanced in LD_B (zeitgeber time 8 [ZT8]; ZT0 representing the onset of darkness) in relation to LD_W and LD_R (ZT11). Under DD conditions, the same rhythm persisted, although with lower amplitude, whereas under LL the hatching rhythm split into two peaks (ZT8 and ZT13). From dph 4 onwards, larvae under LD_B showed the best growth and quickest development (advanced eye pigmentation, mouth opening, and pectoral fins), whereas larvae under LD_R and DD had the poorest performance. These results reveal that developmental rhythms at the egg stage are tightly controlled by light characteristics, underlining the importance of reproducing their natural underwater photoenvironment (LD cycles of blue wavelengths) during incubation and early larvae development of fish. (Author correspondence: borja@um.es)     

Metamorphosis induces a light-dependent switch in Senegalese sole (Solea senegalensis) from diurnal to nocturnal behavior

Light plays a key role in the development of biological rhythms in fish. Recent research in Senegal sole has revealed that spawning and hatching rhythms, larval development, and growth performance are strongly influenced by lighting conditions. However, the effect of light on the daily patterns of behavior remains unexplored. Therefore, the aim of this study was to investigate the impact of different photoperiod regimes and white, blue, and red light on the activity rhythms and foraging behavior of Solea senegalensis larvae up to 40 days posthatching (DPH). To this end, eggs were collected immediately after spawning during the night and exposed to continuous white light (LL), continuous darkness (DD), or light-dark (LD) 12L:12D cycles of white (LD(W)), blue (LD(B), λ(peak) = 463 nm), or red light (LD(R), λ(peak) = 685 nm). A filming scenario was designed to video record activity rhythms during day and night times using infrared lights. The results revealed that activity rhythms in LD(B) and LD(W) changed from diurnal to nocturnal on days 9 to 10 DPH, coinciding with the onset of metamorphosis. In LD(R), sole larvae remained nocturnal throughout the experimental period, while under LL and DD, larvae failed to show any rhythm. In addition, larvae exposed to LD(B) and LD(W) had the highest prey capture success rate (LD(B) = 82.6% ± 2.0%; LD(W) = 75.1% ± 1.3%) and attack rate (LD(B) = 54.3% ± 1.9%; LD(W) = 46.9% ± 3.0%) during the light phase (ML) until 9 DPH. During metamorphosis, the attack and capture success rates in these light conditions were higher during the dark phase (MD), when they showed the same nocturnal behavioral pattern as under LD(R) conditions. These results revealed that the development of sole larvae is tightly controlled by light characteristics, underlining the importance of the natural underwater photoenvironment (LD cycles of blue wavelengths) for the normal onset of the rhythmic behavior of fish larvae during early ontogenesis.     

Effects of constant darkness and constant light on circadian organization and reproductive responses in the ram

The relationship between circadian rhythms in the blood plasma concentrations of melatonin and rhythms in locomotor activity was studied in adult male sheep (Soay rams) exposed to 16-week periods of short days (8 hr of light and 16 hr of darkness; LD 8:16) or long days (LD 16:8) followed by 16-week periods of constant darkness (dim red light; DD) or constant light (LL). Under both LD 8:16 and LD 16:8, there was a clearly defined 24-hr rhythm in plasma concentrations of melatonin, with high levels throughout the dark phase. Periodogram analysis revealed a 24-hr rhythm in locomotor activity under LD 8:16 and LD 16:8. The main bouts of activity occurred during the light phase. A change from LD 8:16 to LD 16:8 resulted in a decrease in the duration of elevated melatonin secretion (melatonin peak) and an increase in the duration of activity corresponding to the changes in the ratio of light to darkness. In all rams, a significant circadian rhythm of activity persisted over the first 2 weeks following transfer from an entraining photoperiod to DD, with a mean period of 23.77 hr. However, the activity rhythms subsequently became disorganized, as did the 24-hr melatonin rhythms. The introduction of a 1-hr light pulse every 24 hr (LD 1:23) for 2 weeks after 8 weeks under DD reinduced a rhythm in both melatonin secretion and activity: the end of the 1-hr light period acted as the dusk signal, producing a normal temporal association of the two rhythms. Under LL, the 24-hr melatonin rhythms were disrupted, though several rams still showed periods of elevated melatonin secretion. Significant activity rhythms were either absent or a weak component occurred with a period of 24 hr. The introduction of a 1-hr dark period every 24 hr for 2 weeks after 8 weeks under LL (LD 23:1) failed to induce or entrain rhythms in either of the parameters. The occurrence of 24-hr activity rhythm in some rams under LL may indicate nonphotoperiodic entrainment signals in our experimental facility. Reproductive responses to the changes in photoperiod were also monitored. After pretreatment with LD 8:16, the rams were sexually active; exposure to LD 16:8, DD, or LL resulted in a decline in all measures of reproductive function. The decline was slower under DD than LD 16:8 or LL.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of Constant Light and Darkness,Light Intensity,and Light Spectrum on Plasma Melatonin Rhythms in Senegal Sole

Light is the most important synchronizer of melatonin rhythms in fish. This paper studies the influence of the characteristics of light on plasma melatonin rhythms in sole. The results revealed that under long‐term exposure to constant light conditions (LL or DD), the total 24 h melatonin production was significantly higher than under LD, but LL and DD conditions influenced the rhythms differently. Under LL, melatonin remained at around 224 pg/ml throughout the 24 h, while under DD a significant elevation (363.6 pg/ml) was observed around the subjective evening. Exposure to 1 h light pulses at MD (mid‐dark) inhibited melatonin production depending on light intensity (3.3, 5.3, 10.3, and 51.9 µW/cm²). The light threshold required to reduce nocturnal plasma melatonin to ML (mid‐light) values was 5.3 µW/cm². Melatonin inhibition by light also depended on the wavelength of the light pulses: while a deep red light (λ>600 nm) failed to reduce plasma melatonin significantly, far violet light (λ_max=368 nm) decreased indoleamine's concentration to ML values. These results suggest that dim light at night (e.g., moonlight) may be perceived and hence affect melatonin rhythms, encouraging synchronization to the lunar cycle. On the other hand, deep red light does not seem to inhibit nocturnal melatonin production, and so it may be used safely during sampling at night.     

Zebrafish Temperature Selection and Synchronization of Locomotor Activity Circadian Rhythm to Ahemeral Cycles of Light and Temperature

In addition to light cycles, temperature cycles are among the most important synchronizers in nature. Indeed, both clock gene expression and circadian activity rhythms entrain to thermocycles. This study aimed to extend our knowledge of the relative strength of light and temperature as zeitgebers for zebrafish locomotor activity rhythms. When the capacity of a 24∶20°C (thermophase∶cryophase, referred to as TC) thermocycle to synchronize activity rhythms under LL was evaluated, it was found that most groups (78%) synchronized to these conditions. Under LD, when zebrafish were allowed to select the water temperature (24°C vs. 20°C), most fish selected the higher temperature and showed diurnal activity, while a small (25%) percentage of fish that preferred the lower temperature displayed nocturnal activity. Under conflicting LD and TC cycles, fish showed diurnal activity when the zeitgebers were in phase or in antiphase, with a high percentage of activity displayed around dawn and dusk (22% and 34% of the total activity for LD/TC and LD/CT, respectively). Finally, to test the relative strength of each zeitgeber, fish were subjected to ahemeral cycles of light (T=25 h) and temperature (T=23 h). Zebrafish synchronized mostly to the light cycle, although they displayed relative coordination, as their locomotor activity increased when light and thermophase coincided. These findings show that although light is a stronger synchronizer than temperature, TC cycles alone can entrain circadian rhythms and interfere in their light synchronization, suggesting the existence of both light‐ and temperature‐entrainable oscillators that are weakly coupled.     

Locomotor activity rhythm in four wrasse species under varying light conditions

Under controlled laboratory conditions, the locomotor activity rhythms of four species of wrasses (Suezichthys gracilis, Thalassoma cupido, Labroides dimidiatus andCirrhilabrus temminckii) were individually examined using an actograph with infra-red photo-electric switches in a dark room at temperatures of 21.3–24.3°C, for 7 to 14 days. The locomotor activity ofS. gracilis occurred mostly during the light period under a light-dark cycle regimen (LD 12:12; 06:00-18:00 light, 18:00-06:00 dark). The locomotor activity commenced at the beginning of the light period and continued until a little before the beginning of dark period. The diel activity rhythm of this species synchronizes with LD. Under constant illumination (LL) this species shows distinct free-running activity rhythms varying in length from 23 hrs. 39 min. to 23 hrs. 47 min. Therefore,S. gracilis appears to have a circadian rhythm under LL. However, in constant darkness (DD), the activity of this species was greatly suppressed. All the fish showed no activity rhythms in DD conditions. After DD, the fish showed the diel activity rhythm with the resumption of LD, but this activity began shortly after the beginning of light period. The fish required several days to synchronize with the activity in the light period. Therefore,S. gracilis appeared to continue the circadian rhythm under DD. InT. cupido, the locomotor activity commenced somewhat earlier than the beginning of the light period and continued until the beginning of the dark period under LD. The diel activity rhythm of this species synchronizes with LD. Under LL, four of the five specimens of this species tested showed free-running activity rhythms for the first 5 days or longer varying in length from 22 hrs. 54 min. to 23 hrs. 39 min. Although the activity of this species was suppressed under DD, two of five fish showed free-running activity rhythms throughout the experimental period. The lengths of such free-running periods were from 23 hrs. 38 min. to 23 hrs. 50 min. under DD. Therefore, it was ascertained thatT. cupido has a circadian rhythm. InL. dimidiatus, the locomotor activity rhythm under LD resembled that observed inT. cupido. The diel activity rhythm of this species synchronizes with LD. Under LL, four of seven of this species showed free-running activity rhythms throughout the experimental period. The lengths of such free-running periods were from 23 hrs. 07 min. to 25 hrs. 48 min. Although the activity of this species was suppressed under DD, three of five fish showed free-running activity rhythms throughout the experimental period. The lengths of such free-running periods were from 23 hrs. 36 min. to 23 hrs. 41 min. under DD. Therefore, it was ascertained thatL. dimidiatus has a circadian rhythm. Almost all locomotor activity of C.temminckii occurred during the light period under LD. The diel activity rhythm of this species coincides with LD. Under LL, two of four of this species showed free-running activity rhythms throughout the experimental period. The lengths of such free-running periods were from 23 hrs. 32 min. to 23 hrs. 45 min. Although the activity of this species was suppressed under DD, one of the four fish showed free-running activity rhythms throughout the experimental period. The length of the free-running period was 23 hrs. 21 min. under DD. Therefore,C. temminckii appeared to have a circadian rhythm. According to field observations,S. gracilis burrows and lies in the sandy bottom whileT. cupido, L. dimidiatus, andC. temminckii hide and rest in spaces among piles of boulders or in crevices of rocks during the night. It seems that the differences in nocturnal behavior among the four species of wrasses mentioned above are closely related to the intensity of endogenous factors in their locomotor activity rhythms.     

Light and temperature cycles as zeitgebers of zebrafish (Danio rerio) circadian activity rhythms

Light and temperature cycles are the most important synchronizers of biological rhythms in nature. However, the relative importance of each, especially when they are not in phase, has been poorly studied. The aim of this study was to analyze the entrainment of daily locomotor activity to light and/or temperature cycles in zebrafish. Under two constant temperatures (20°C and 26°C) and 12:12 light-dark (LD) cycles, zebrafish were most active during the day (light) time and showed higher total activity at the warmer temperature, while diurnalism was higher at 20°C than at 26°C (87% and 77%, respectively). Under thermocycles (12:12 LD, 26:20°C thermophase:chryophase or TC), zebrafish daily activity synchronized to the light phase, both when the thermophase and light phase were in phase (LD/TC) or in antiphase (LD/CT). Under constant dim light (3 lux), nearly all zebrafish synchronized to thermocycles (τ=24 h), although activity rhythms (60% to 67% of activity occurred during the thermophase) were not as marked as those observed under the LD cycle. Under constant dim light of 3 lux and constant temperature (22.5°C), 4 of 6 groups of zebrafish previously entrained to thermocycles displayed free-running rhythms (τ=22.9 to 23.6 h). These results indicate that temperature cycles alone can also entrain zebrafish locomotor activity.     

Impact of Daily Thermocycles on Hatching Rhythms,Larval Performance and Sex Differentiation of Zebrafish

In the wild, water temperature cycles daily: it warms up after sunrise, and cools rapidly after sunset. Surprisingly, the impact of such daily thermocycles during the early development of fish remains neglected. We investigated the influence of constant vs daily thermocycles in zebrafish, from embryo development to sexual differentiation, by applying four temperature regimens: two constant (24°C and 28°C) and two daily thermocycles: 28:24°C, TC (thermophase coinciding with daytime, and cryophase coinciding with night-time) and 24:28°C, CT (opposite to TC) in a 12:12 h light:dark cycle (LD). Embryo development was temperature-dependent but enhanced at 28°C and TC. Hatching rhythms were diurnal (around 4 h after lights on), but temperature- and cycle-sensitive, since hatching occurred sooner at 28°C (48 hours post fertilization; hpf) while it was delayed at 24°C (96 hpf). Under TC, hatching occurred at 72 hpf, while under CT hatching displayed two peaks (at 70 hpf and 94 hpf). In constant light (LL) or darkness (DD), hatching rhythms persisted with tau close to 24 h, suggesting a clock-controlled “gating” mechanism. Under 28°C or TC, larvae showed the best performance (high growth and survival, and low malformations). The sex ratio was strongly influenced by temperature, as the proportion of females was higher in CT and TC (79 and 83% respectively), contrasting with 28°C and 24°C, which led to more males (83 and 76%). Ovarian aromatase (cyp19a) expression in females was highest in TC and CT (6.5 and 4.6 fold higher than at 28°C, respectively); while anti-müllerian hormone (amh) expression in males increased in testis at 24°C (3.6 fold higher compared to TC) and particularly at 28°C (14.3 fold increase). Taken together, these findings highlight the key role of environmental cycles during early development, which shaped the daily rhythms in fish embryo and larvae, and ultimately influenced sex differentiation.     

The relationship between locomotor activity rhythm and burying behavior in the wrasse,Suezichthys gracilis

The wrasse,Suezichthys gracilis, is a diurnal fish which buries itself in sand during the night-time. The present paper deals with the locomotor activity rhythms ofS. gracilis, examined by using an actograph with infra-red photo-electric switches in a dark room. The fish were kept in eight experimental tanks (each 30l in capacity), with three different bottom conditions: sand (grain size about 1 mm in diameter and 5 cm deep); 1 or 2 stones (about 10cm in diameter) without sand; and transparent acrylic pellets (2 × 2 × 3 mm in size, 5 cm deep). The light intensities were 550–700 lux just above the water surface, decreasing to 21.3% under the acrylic pellets at a water depth of 20cm. The water temperatures were kept at 22.0–25.0°C during the experiments for 7 to 14 days. In the aquarium with bottom sand, diel activity rhythms ofS. gracilis were mostly synchronized to LD (LD12:12; 06:00–18:00 light, 18:00–06:00 dark), free-running activity rhythms continued distinctly under LL (constant illumination), and locomotor activity was greatly suppressed, with disappearance of the activity rhythm, under DD (constant darkness). In the aquarium without sand, locomotor activity ofS. gracilis could be summarized as follows. The fish moved throughout almost the entire period under LD, though more frequent movements were observed in light conditions than in dark ones. Under LL they showed continuous locomotor activity during the experiment, with no obvious periodicity. Under DD the activity of the species was somewhat suppressed, but irregular movement or indistinct periodicity was observed. In the aquarium with transparent acrylic pellets, locomotor activity under LD and DD, respectively, bore a close resemblance to activity patterns under the same light conditions with sand, whilst activity under LL was identical to that under LL without sand. Accordingly, it seems that maintenance of normal activity rhythms in the wrasse was due not only to the darkness, but also to the presence of bottom sand. It therefore seems that the biological clock inS. gracilis is not related to locomotor activity, but to burying behavior.     

Influence of constant light and darkness, light intensity, and light spectrum on plasma melatonin rhythms in senegal sole

Light is the most important synchronizer of melatonin rhythms in fish. This paper studies the influence of the characteristics of light on plasma melatonin rhythms in sole. The results revealed that under long-term exposure to constant light conditions (LL or DD), the total 24 h melatonin production was significantly higher than under LD, but LL and DD conditions influenced the rhythms differently. Under LL, melatonin remained at around 224 pg/ml throughout the 24 h, while under DD a significant elevation (363.6 pg/ml) was observed around the subjective evening. Exposure to 1 h light pulses at MD (mid-dark) inhibited melatonin production depending on light intensity (3.3, 5.3, 10.3, and 51.9 microW/cm(2)). The light threshold required to reduce nocturnal plasma melatonin to ML (mid-light) values was 5.3 microW/cm(2). Melatonin inhibition by light also depended on the wavelength of the light pulses: while a deep red light (lambda>600 nm) failed to reduce plasma melatonin significantly, far violet light (lambda(max)=368 nm) decreased indoleamine's concentration to ML values. These results suggest that dim light at night (e.g., moonlight) may be perceived and hence affect melatonin rhythms, encouraging synchronization to the lunar cycle. On the other hand, deep red light does not seem to inhibit nocturnal melatonin production, and so it may be used safely during sampling at night.     

Circadian organization of a subarctic rodent, the northern red-backed vole (Clethrionomys rutilus)

Arctic and subarctic environments are exposed to extreme light: dark (LD) regimes, including periods of constant light (LL) and constant dark (DD) and large daily changes in day length, but very little is known about circadian rhythms of mammals at high latitudes. The authors investigated the circadian rhythms of a subarctic population of northern red-backed voles (Clethrionomys rutilus). Both wild-caught and third-generation laboratory-bred animals showed predominantly nocturnal patterns of wheel running when exposed to a 16:8 LD cycle. In LL and DD conditions, animals displayed large phenotypic variation in circadian rhythms. Compared to wheel-running rhythms under a 16:8 LD cycle, the robustness of circadian activity rhythms decreased among all animals tested in LL and DD (i.e., decreased chi-squared periodogram waveform amplitude). A large segment of the population became noncircadian (60% in DD, 72% in LL) within 8 weeks of exposure to constant lighting conditions, of which the majority became ultradian, with a few individuals becoming arrhythmic, indicating highly labile circadian organization. Wild-caught and laboratory-bred animals that remained circadian in wheel running displayed free-running periods between 23.3 and 24.8 h. A phase-response curve to light pulses in DD showed significant phase delays at circadian times 12 and 15, indicating the capacity to entrain to rapidly changing day lengths at high latitudes. Whether this phenotypic variation in circadian organization, with circadian, ultradian, and arrhythmic wheel-running activity patterns in constant lighting conditions, is a novel adaptation to life in the arctic remains to be elucidated.     

Thermoperiodic response and effect of photoperiod on thermoperiodic induction of diapause in Colaphellus bowringi

The effects of thermoperiods on diapause induction under continuous darkness (DD), continuous light (LL), and an L12:D12 photoperiod were investigated in the cabbage beetle, Colaphellus bowringi Baly (Coleoptera: Chrysomelidae), a short‐day species. Diapause could be induced by thermoperiod under both LL and DD; however, in the range of 24–30 °C, lower incidences of diapause were observed under LL than under DD. The critical cryophase was found to be dependent on the mean temperature of the thermoperiod applied. Although the thermoperiodic response pattern was similar under LL and DD, the incidence of diapause was higher under LL when the duration of the cryophase did not exceed 12 h. In contrast, when the duration of the cryophase was longer than 12 h, the incidence of diapause under LL was lower or equal to that under DD. When a thermoperiod of 24 °C (cryophase) and 28 °C (thermophase) was applied, the incidence of diapause was higher under LL than under DD, regardless of the duration of the cryophase. Thermoperiodic responses under a photoperiod of L12:D12 and under DD further revealed that induction of diapause was strongly influenced by the photophase temperature. Moreover, the incidence of diapause was lower when the thermophase coincided with the photophase than when the cryophase coincided with the photophase.     

The Circadian Clock Machinery During Early Development of Senegalese sole (Solea senegalensis): Effects of Constant Light and Dark Conditions

Circadian rhythms are established very early during vertebrate development. In fish, environmental cues can influence the initiation and synchronization of different rhythmic processes. Previous studies in zebrafish and rainbow trout have shown that circadian oscillation of clock genes represents one of the earliest detectable rhythms in the developing embryo, suggesting their significance in regulating the coordination of developmental processes. In this study, we analyzed the daily expression of the core clock components Per1, Per2, Per3, and Clock during the first several days of Senegalese sole development (0–4 d post fertilization or dpf) under different lighting regimes, with the aim of addressing when the molecular clock first emerges in this species and how it is affected by different photoperiods. Rhythmic expression of the above genes was detected from 0 to 1 dpf, being markedly affected in the next few days by both constant light (LL) and dark (DD) conditions. A gradual entrainment of the clock machinery was observed only under light-dark (LD) cycles, and robust rhythms with increased amplitudes were established by 4 dpf for all clock genes currently studied. Our results show the existence of an embryonic molecular clock from the 1st d of development in Senegalese sole and emphasize the significance of cycling LD conditions when raising embryos and early larvae. (Author correspondence: munoz.cueto@uca.es; carlos.pendon@uca.es)     

Circadian Intraocular Pressure Rhythms in Athletic Horses under Different Lighting Regime

The present study was undertaken to investigate the existence of intraocular pressure (IOP) rhythms in athletic thoroughbred horses maintained under a 24 h cycle of light and darkness (LD) or under constant light (LL) or constant dark (DD) conditions. We identified an IOP circadian rhythm that is entrained to the 24 h LD cycle. IOP was low during the dark phase and high during the light phase, with a peak at the end of the light phase (ZT10). The circadian rhythm of IOP persisted in DD (with a peak at CT9.5), demonstrating an endogenous component in IOP rhythm. As previously shown in other mammalian species, horse IOP circadian rhythmicity was abolished in LL. Because tonometry is performed in horses for the diagnosis of ophthalmologic diseases, such as glaucoma or anterior uveitis, the daily variation in IOP must be taken into account in clinical practice to properly time tests and to interpret clinical findings.     

Ontogeny of the circadian system governing ecdysial rhythms in a holometabolous insect, Diatraea grandiosella (Pyralidae)

ABSTRACT. Three eclosion rhythms of Diatraea grandiosella Dyar (Lepidoptera, Pyralidae) showed different phase relationships to the LD cycle, different degrees of synchronization and different sensitivity to a brief light perturbation. Egg hatching occurred mainly at dawn, whereas pupation and adult emergence occurred mainly at dusk. The egg hatching was the most synchronized rhythm of the three, pupation the least. The egg hatching rhythm was more sensitive to a brief light perturbation than was the adult emergence rhythm. A light pulse in the middle of the night caused greater disturbance in the egg hatching rhythm than did a pulse placed in the early night. The three rhythms persisted in both DD and LL. LL free-running rhythm gradually became desynchronized and broke down at the third cycle. The phase relationships reveal difficulties in applying the 'external coincidence model' to this species' photoperiodic response.     

Daily rhythms of clock gene expression and feeding behavior during the larval development in gilthead seabream,Sparus aurata

Light is the main environmental time cue which synchronizes daily rhythms and the molecular clock of vertebrates. Indeed, alterations in photoperiod have profound physiological effects in fish (e.g. reproduction and early development). In order to identify the changes in clock genes expression in gilthead seabream larvae during ontogeny, three different photoperiods were tested: a regular 12L:12D cycle (LD), a continuous light 24L:0D (LL) and a two-phases photoperiod (LL?+?LD) in which the photoperiod changed from LL to LD on day 15 after hatching (dph). Larvae were sampled on 10, 18, 30 and 60 days post-hatch (dph) during a 24?h cycle. In addition to the expression of clock genes (clock, bmal1, cry1 and per3), food intake was measured. Under LD photoperiod, larvae feed intake and clock genes expression showed a rhythmic pattern with a strong light synchronization, with the acrophases occurring at the same hour in all tested ages. Under LL photoperiod, the larvae also showed a rhythmic pattern but the acrophases occurred at different times depending on the age, although at the end of the experiment (60 dph) clock genes expression and feed intake rhythms were similar to those larvae exposed to LD photoperiod. Moreover, the expression levels of bmal1 and cry1 were much lower than in LD photoperiod. Under the LL?+?LD photoperiod, the 10 dph larvae showed the same patterns as LL treatment while 18 and 30 dph larvae showed the same patterns as LD treatment. These results revealed the presence of internal factors driving rhythmic physiological responses during larvae development under constant environmental conditions. The LL?+?LD treatment demonstrates the plasticity of the clock genes expression and the strong effect of light as synchronizer in developing fish larvae.     

Circadian Pattern of Wheel‐Running Activity of a South American Subterranean Rodent (Ctenomys cf knightii)

Circadian rhythms are regarded as essentially ubiquitous features of animal behavior and are thought to confer important adaptive advantages. However, although circadian systems of rodents have been among the most extensively studied, most comparative biology is restricted to a few related species. In this study, the circadian organization of locomotor activity was studied in the subterranean, solitary north Argentinean rodent, Ctenomys knightii. The genus, Ctenomys, commonly known as Tuco‐tucos, comprises more than 50 known species over a range that extends from 12°S latitude into Patagonia, and includes at least one social species. The genus, therefore, is ideal for comparative and ecological studies of circadian rhythms. Ctenomys knightii is the first of these to be studied for its circadian behavior. All animals were wild caught but adapted quickly to laboratory conditions, with clear and precise activity‐rest rhythms in a light‐dark (LD) cycle and strongly nocturnal wheel running behavior. In constant dark (DD), the rhythm expression persisted with free‐running periods always longer than 24 h. Upon reinstatement of the LD cycle, rhythms resynchronized rapidly with large phase advances in 7/8 animals. In constant light (LL), six animals had free‐running periods shorter than in DD, and 4/8 showed evidence of “splitting.” We conclude that under laboratory conditions, in wheel‐running cages, this species shows a clear nocturnal rhythmic organization controlled by an endogenous circadian oscillator that is entrained to 24 h LD cycles, predominantly by light‐induced advances, and shows the same interindividual variable responses to constant light as reported in other non‐subterranean species. These data are the first step toward understanding the chronobiology of the largest genus of subterranean rodents.     

Effects of background light conditions on thermoperiodic eclosion rhythm of onion fly Delia antiqua

To elucidate the effects of light on thermoperiodic regulation of adult eclosion rhythm in the onion fly, Delia antiqua, the responses to two thermoperiods, 29°C (12 h):21°C (12 h) and 25.5°C (12 h):24.5°C (12 h), with different amplitude and same average temperature, were examined in continuous darkness (DD) and continuous light (LL). Irrespective of the temperature step between warm phase (W) and cool phase (C), temperature cycles effectively entrained the adult eclosion rhythm in both DD and LL. Eclosion peaks, however, varied with light conditions and temperature step between W and C. It advanced by approximately 2–3 h in DD than in LL and at smaller temperature step. Background light conditions and temperature step also affect the amplitude of eclosion rhythm. It became lower in LL than in DD and at smaller temperature steps. On transfer to constant temperature (25°C), eclosion rhythm was elicited earliest in the pupae at 8°C temperature step in DD and latest in those at 1°C temperature step in LL. Pupae at 1°C temperature step in DD and at 8°C temperature step in LL demonstrated intermediate responses, but the eclosion rhythm was elicited 1 day earlier in the former than in the latter. This might be ascribed to the interaction between background light and temperature step under thermoperiodic conditions. The results suggest that continuous light and a smaller temperature step weaken the coupling strength between eclosion rhythm and thermoperiod, but the light effect is stronger than the temperature step effect.