24-h Light-dark Research Articles

Postnatal light alters hypothalamic-pituitary-adrenal axis function and induces a depressive-like phenotype in adult mice.

The postnatal light environment that a mouse experiences during the critical first three postnatal weeks has long-term effects on both its circadian rhythm output and clock gene expression. Furthermore, data from our lab suggest that postnatal light may also impact the hypothalamic-pituitary-adrenal (HPA) axis, which is a key regulator of stress. To test the effect of postnatal light exposure on adult stress responses and circadian rhythmicity, we raised mice under either 24-h light-dark cycles (LD), constant light (LL) or constant dark (DD) during the first three postnatal weeks. After weaning we then exposed all animals to LD cycles (basal conditions), followed by LL (stressed conditions) environments. We examined brain neuropeptide and glucocorticoid receptor (GR) expression, plasma corticosterone concentration rhythm and body temperature rhythm, together with depression- and anxiety-related behaviour. Results showed that LL- and DD-raised mice exhibited decreased GR expression in the hippocampus, increased plasma corticosterone concentration at the onset of the dark phase and a depressive phenotype when exposed to LD cycles later in life. Furthermore, LL-raised mice showed increased corticotrophin-releasing hormone mRNA expression in the paraventricular nucleus of the hypothalamus. When exposed to LL as adults, LL-raised mice showed a significant circadian rhythm of plasma corticosterone concentration, together with a shorter period and stronger circadian rhythm of body temperature compared to DD-raised mice. Taken together, these data suggest that altered postnatal light environments have long-term effects on the HPA axis and the circadian system, which can lead to altered stress responses and a depressive phenotype in adulthood.

Mir-276a strengthens Drosophila circadian rhythms by regulating timeless expression

Circadian rhythms in metazoan eukaryotes are controlled by an endogenous molecular clock. It functions in many locations, including subsets of brain neurons (clock neurons) within the central nervous system. Although the molecular clock relies on transcription/translation feedback loops, posttranscriptional regulation also plays an important role. Here, we show that the abundant Drosophila melanogaster microRNA mir-276a regulates molecular and behavioral rhythms by inhibiting expression of the important clock gene timeless (tim). Misregulation of mir-276a in clock neurons alters tim expression and increases arrhythmicity under standard constant darkness (DD) conditions. mir-276a expression itself appears to be light-regulated because its levels oscillate under 24-h light-dark (LD) cycles but not in DD. mir-276a is regulated by the transcription activator Chorion factor 2 in flies and in tissue-culture cells. Evidence from flies mutated using the clustered, regularly interspaced, short palindromic repeats (CRISPR) tool shows that mir-276a inhibits tim expression: Deleting the mir-276a-binding site in the tim 3' UTR causes elevated levels of TIM and ∼50% arrhythmicity. We suggest that this pathway contributes to the more robust rhythms observed under light/dark LD conditions than under DD conditions.

Characterization of the relationship between spontaneous locomotor activity and cardiovascular parameters in conscious freely moving rats

In freely behaving rats, variations in heart rate (HR) and blood pressure (BP) are coupled closely with changes in locomotor activity (Act). We have attempted to characterize this relationship mathematically. In 10- and 16-week-old rats, HR, BP and Act were recorded telemetrically every minute for 2days under 12h:12h light–dark cycling. After examining data for individual rats, we found that the relationship between Act and HR could be approximated by the negative exponential function HR(Act)=HRmax−(HRmax−HRmin)∗exp(−Act/Acte), where HRmax, HRmin, and Acte are constants. These constants were calculated separately for light and dark periods by non-linear curve fitting. HR corresponding to maximal locomotion was similar during the light and dark phases, while HR at rest during the dark phase was higher than during the light phase. The range of HR variability associated with Act during the dark phase was similar in young and older animals, but minimal HR was significantly lower in older rats. The relationship between Act and BP was approximated with a similar function. We have found no differences between BP at rest and at maximal locomotion between light and dark and between 10-week and 16-week-old rats. Our results indicate that in rats, cardiovascular parameters are coupled to locomotion to a high degree; however both the HR and the BP reach maximal values when locomotor activity is relatively low. We also found that the phase of daily cycle affects HR in conscious rats independent of locomotor activity.

Control of activity patterns in crowded groups of male noble crayfish Astacus astacus (Crustacea, Astacidea) by light regimes: A way to increase the efficiency of crayfish production?

Abstract Most crayfish species exhibit behavioral traits (solitary lifestyle, high aggressiveness, establishment of social hierarchies) which in intensive aquaculture systems result in low survival due to cannibalism and reduced overall growth. In addition to selective breeding programs, this situation may be improved by identifying culture conditions (e.g., light regimes) which can alleviate the negative impact of specific behavioral patterns on an effective crayfish production. The present experiments demonstrate a pronounced nocturnality of male noble crayfish Astacus astacus. The circadian system seems to be of rather little relevance to the daily pattern of the collective activity in crowded crayfish groups. Parameters of the light regime (the L/D-ratio of 24-h light–dark cycles, light intensity, background illumination during nighttime) were shown to have a crucial and immediate influence on (i) the daily temporal pattern of activity, (ii) the ‘effective density’ (density of individuals which are simultaneously active), and (iii) the overall daily amount of activity. Except for individuals at the verge of molting, activities were restricted to the scotophase, largely independent of the LD-ratio. The effective density was negatively, the overall daily amount of activity positively, correlated with the length of the scotophase. Both parameters decreased significantly under a dim nocturnal background illumination. An optimal combination of high overall daily activity and low effective density was found under a short-day photoperiod of LD 8:16. As in the long run overall daily amount of activity and effective density may be relevant to growth and survival, respectively, manipulations of the light regime are suggested to be a promising approach to improve the efficiency of a commercial crayfish production. The present results may be useful in designing appropriate long-term experiments which actually measure survival and growth. Statement of relevance Light as potential factor to improve production in RASs.

Day and night variations in the repair of ionizing-radiation-induced DNA damage in mouse splenocytes.

In mammals, biological rhythms synchronize physiological and behavioral processes to the 24-h light-dark (LD) cycle. At the molecular level, self-sustaining processes, such as oscillations of transcription-translation feedback loops, control the circadian clock, which in turn regulates a wide variety of cellular processes, including gene expression and cell cycle progression. Furthermore, previous studies reported circadian oscillations in the repair capacity of DNA lesions specifically repaired by nucleotide excision repair (NER). However, it is so far only poorly understood if DNA repair pathways other than NER are under circadian control, in particular base excision and DNA strand break repair. In the present study, we analyzed potential day and night variations in the repair of DNA lesions induced by ionizing radiation (i.e., mainly oxidative damage and DNA strand breaks) in living mouse splenocytes using a modified protocol of the automated FADU assay. Our results reveal that splenocytes isolated from mice during the light phase (ZT06) displayed higher DNA repair activity than those of the dark phase (ZT18). As analyzed by highly sensitive and accurate qPCR arrays, these alterations were accompanied by significant differences in expression profiles of genes involved in the circadian clock and DNA repair. Notably, the majority of the DNA repair genes were expressed at higher levels during the light phase (ZT06). This included genes of all major DNA repair pathways with the strongest differences observed for genes of base excision and DNA double strand break repair. In conclusion, here we provide novel evidence that mouse splenocytes exhibit significant differences in the repair of IR-induced DNA damage during the LD cycle, both on a functional and on a gene expression level. It will be interesting to test if these findings could be exploited for therapeutic purposes, e.g. time-of-the-day-specific application of DNA-damaging treatments used against blood malignancies.

Effect of salinity and light intensity on somatic growth and podocyst production in polyps of the giant jellyfish Nemopilema nomurai (Scyphozoa: Rhizostomeae)

Frequent blooms of the giant jellyfish, Nemopilema nomurai, have occurred in the East Asian Marginal Seas since 2000 and have had a devastating effect on commercial fisheries. To determine the probability of future bloom development to permit effective countermeasures, it is essential to assess the influence of environmental factors on polyp development. In this study, the effects of salinity on the polyps of N. nomurai from the coastal waters of Dalian in the northern Yellow Sea were tested at salinities ranging from 2.5 to 45. The combined effects of salinity (15 and 32) and light intensity (800, 400, and 0 lux) in a 12-h light–12-h dark photoperiod were also assessed. The lower and upper salinity limits for N. nomurai polyp survival were 10 and 40, respectively. The salinity range of 20–27.5 was best for podocyst reproduction. Survival and somatic growth of polyps, and their asexual production of podocysts, were significantly greater at low to middle than at high salinities. Polyps survived, grew, and produced podocysts at all light intensities tested; however, differences in light intensity induced few statistically significant changes. These results indicated that N. nomurai polyps are adapted to survive, grow, and reproduce in estuaries and near-shore environments.

Sensitivity of housekeeping genes in the suprachiasmatic nucleus of the mouse brain to diet and the daily light–dark cycle

The endogenous timing system within the suprachiasmatic nuclei (SCN) of the hypothalamus drives the cyclic expression of the clock molecules across the 24h day–night cycle controlling downstream molecular pathways and physiological processes. The developing fetal clock system is sensitive to the environment and physiology of the pregnant mother and as such disruption of this system could lead to altered physiology in the offspring. Characterizing the gene profiles of the endogenous molecular clock system by quantitative reverse transcription polymerase chain reaction is dependent on normalization by appropriate housekeeping genes (HKGs). However, many HKGs commonly used as internal controls, although stably expressed under control conditions, can vary significantly in their expression under certain experimental conditions. Here we analyzed the expression of 10 classic HKG across the 24h light–dark cycle in the SCN of mouse offspring exposed to normal chow or a high fat diet during early development and in postnatal life. We found that the HKGs glyceraldehyde-3-phosphate dehydrogenase, beta actin and adenosine triphosphate synthase subunit to be the most stably expressed genes in the SCN regardless of diet or time within the 24h light–dark cycle, and are therefore suitable to be used as internal controls. However SCN samples collected during the light and dark periods did show differences in expression and as such the timing of collection should be considered when carrying out gene expression studies.

Effect of Lighting Conditions on Zebrafish Growth and Development

In the underwater environment, the properties of light (intensity and spectrum) change rapidly with depth and water quality. In this article, we have described how and to what extent lighting conditions can influence the development, growth, and survival of zebrafish. Fertilized eggs and the corresponding larvae were exposed to different visible light wavelengths (violet, blue, green, yellow, red, and white) in a 12-h light-12-h dark (LD) cycle until 30 days posthatching (dph), when the expression of morphometric parameters and growth (igf1a, igf2a)- and stress-related (crh and pomca) genes were examined. Another group of larvae was raised under constant darkness (DD) until 5 or 10 dph, after which they were transferred to a LD of white light. A third group remained under DD to investigate the effects of light deprivation upon zebrafish development. The results revealed that the hatching rate was highest under blue and violet light, while total length at 30 dph was greatest under blue, white, and violet light. Red light led to reduced feeding activity and poor survival (100% mortality). Larvae raised under constant white light (LL) showed a higher proportion of malformations, as did larvae raised under LD violet light. The expression of growth and stress factors was upregulated in the violet (igf1a, igf2a, pomca, and chr) and blue (igf2a) groups, which is consistent with the higher growth recorded and the higher proportion of malformations detected under the violet light. All larvae kept under DD died before 18 dph, but the survival rates improved in larvae transferred to LD at 5 dph and at 10 dph. In summary, these findings revealed that lighting conditions are crucial factors influencing zebrafish larval development and growth.

Differential effects of retinal degeneration on sleep and wakefulness responses to short light–dark cycles in albino mice

This study characterizes the different response patterns of sleep and wakefulness (W) to short light–dark (LD) cycles in albino mice and examines whether retinal degeneration resulting from prolonged bright light treatment and/or rd/rd mutation alters such response patterns. Eight young male Institute for Cancer Research (ICR) mice with normal eyes, seven young male rd/rd Friend Virus B type (FVB) mice, six young ICR and five young rd/rd FVB mice receiving 48-h bright light treatment, and five older rd/rd FVB mice were implanted with skull and muscle electrodes to record sleep and W. All the mice were maintained in 12-h–12-h LD cycles at baseline and received 2days of short LD cycle treatment, which included 5-min–5-min LD cycles for a total of 24 cycles presented 4h after lights-on and again 4h after lights-off. All the five mouse groups maintained photo-entrainment of sleep and W rhythms at the baseline and showed a preference for paradoxical sleep (PS) occurrence in the 5-min dark period and non-rapid eye movement sleep (NREMS) in the 5-min light period and a brief alerting effect of light onset on experimental days. Retinal degeneration rising from bright light treatment and/or genetic mutation failed to eliminate or reduce the response of PS and NREMS to short LD cycles, although it enhanced the LD contrast of W, i.e., bright light treatment prolonged the alerting effect of light and the rd mutation increased the suppressing effect of the dark on W. These results suggest that sleep responses to short LD cycles and the brief alerting effect of light were independent of the photoreceptors in the outer retina. Furthermore, the residual photoreceptors in the outer retina and/or the photosensitive cells in the inner retina may actively modulate the effect of light and dark signals on W.

Effects of temperature, light regime and substrates on the production and germination of Stenocarpella maydis pycnidiospores

This study aimed to evaluate the production and germination of Stenocarpella maydis pycnidiospores under in vitro conditions as affected by substrate composition (sorghum, wheat, black oat or barley), light regime (continuous dark, 12-h light dark -1 or continuous light conditions), and incubation temperature (21, 24, 27, 30 or 33oC). Each substrate (20 g of grain) was soaked in 100 mL of water for 24h and sterilized twice for 20 min at 127oC. Three plugs (5 mm diameter) of a single-spored culture of S. maydis were used as inocula for each substrate. Assessments of pycnidiospore production per gram of grain and percent germination were made 14 days after inoculation. Barley, black oat or wheat grains were the best substrates for the mass production of S. maydis pycnidiospores and the maintenance of high germination rates. The highest pycnidiospore production (67,600 pycnidiospores g -1 ) was obtained using barley grain as a substrate with incubation at 27oC under a 12-h light dark -1 cycle.

Differential Regulation of Arylalkylamine N-Acetyltransferase Activity in Chicken Retinal Ganglion Cells by Light and Circadian Clock

Retinal ganglion cells (RGCs) contain circadian clocks driving melatonin synthesis during the day, a subset of these cells acting as nonvisual photoreceptors sending photic information to the brain. In this work, the authors investigated the temporal and light regulation of arylalkylamine N-acetyltransferase (AA-NAT) activity, a key enzyme in melatonin synthesis. The authors first examined this activity in RGCs of wild-type chickens and compared it to that in photoreceptor cells (PRs) from animals maintained for 48 h in constant dark (DD), light (LL), or regular 12-h:12-h light-dark (LD) cycle. AA-NAT activity in RGCs displayed circadian rhythmicity, with highest levels during the subjective day in both DD and LL as well as in the light phase of the LD cycle. In contrast, AA-NAT activity in PRs exhibited the typical nocturnal peak in DD and LD, but no detectable oscillation was observed under LL, under which conditions the levels were basal at all times examined. A light pulse of 30–60 min significantly decreased AA-NAT activity in PRs during the subjective night, but had no effect on RGCs during the day or night. Intraocular injection of dopamine (50 nmol/eye) during the night to mimic the effect of light presented significant inhibition of AA-NAT activity in PRs compared to controls but had no effect on RGCs. The results clearly demonstrate that the regulation of the diurnal increase in AA-NAT activity in RGCs of chickens undergoes a different control mechanism from that observed in PRs, in which the endogenous clock, light, and dopamine exhibited differential effects. (Author correspondence: mguido@fcq.unc.edu.ar)

Exposure to dexamethasone during late gestation causes female-specific decreases in core body temperature and prepro-thyrotropin-releasing hormone expression in the paraventricular nucleus of the hypothalamus in rats

Synthetic glucocorticoids (GC) have been used to promote lung development in preterm infants, thereby decreasing respiratory distress syndrome and mortality, yet, concern has arisen from reports that such treatment predisposes individuals to disease in adulthood. Given the variety of preclinical studies that show metabolic and behavioral abnormalities in adulthood following fetal exposure to synthetic GC, we examined the effect of in utero exposure to the synthetic GC, dexamethasone (DEX), on hypothalamic expression of thyrotropin-releasing hormone (TRH) a central neuropeptide involved in mediating behavior and metabolic balance. Pregnant Sprague–Dawley rats were administered 0.4mg/kg DEX on gestational days 18–21. As adults (postnatal day (PD) 60), the offspring were fitted with temperature sensing transmitters allowing real-time monitoring of core body temperature (CBT) across the 24h light dark period. This revealed a significant decrease in CBT throughout the day in prenatal DEX-treated females on estrus and diestrus, but not in male offspring. The reduction in CBT by prenatal DEX exposure was accompanied by a significant decrease in the expression of Trh transcript in the paraventricular nucleus of the hypothalamus (PVN) of female rats at PD 60 and this effect was also present on PD7. There was also a female-specific reduction in the number of preproTRH-immunoreactive (ir) neurons in the PVN, with ppTRH-ir nerve fibers decreases that were present in both male and female offspring. No changes in thyroid hormone (triiodothyronine, T3; thyroxine, T4) were observed in adult offspring, but during development, both males and females (PD14) had lower T3 and T4 levels. These data indicate abnormal expression of TRH results from fetal DEX exposure during late gestation, possibly explaining the decreased CBT observed in the female offspring.

The effect of photoperiod on larval culture performance of two marine finfish species

Light can have significant impacts on larval growth and survival of many marine fish species. Here, we study the effect of photoperiod on larvae from captive populations of two marine finfish species, California yellowtail (CYT; Seriola lalandi) and white seabass (WSB; Atractoscion nobilis). Two photoperiod treatments were tested: 12h light and 12h dark (12-L), and 24h light and 0h dark (24-L). CYT larvae were significantly heavier (240±16μg dry weight) in the 24-L treatment group than the 12-L group (155±25μg dry weight) at 10dph. Similarly, WSB were significantly heavier (685±77μg dry weight) in the 24-L treatment group than the 12-L group (455±44μg dry weight) at 18dph. CYT larvae also had significantly greater survival (16.6±6.2%) in the 24-L treatment over the 12-L conditions (7.6±4.9%). In contrast, WSB larvae showed no significant difference in survival between treatments. Food consumption was significantly greater in the 24-L treatment for both species up until 6dph, after which time no difference was observed. Swim bladder inflation rates were not significantly affected by the photoperiod treatments. These results indicate that photoperiod is a significant factor in early larval performance for both CYT and WSB, although it is unknown if continuous light becomes detrimental as larvae develop.

Visible light increases locomotor activity but does not influence body orientation or crawling direction of the nudibranch mollusk Tritonia diomedea

Visible light increases locomotor activity but does not influence body orientation or crawling direction of the nudibranch mollusk Tritonia diomedea

Measurements of Light at Night (LAN) for a Sample of Female School Teachers

Epidemiological studies have shown an association between rotating shiftwork and breast cancer (BC) risk. Recently, light at night (LAN) measured by satellite photometry and by self-reports of bedroom brightness has been shown to be associated with BC risk, irrespective of shiftwork history. Importance has been placed on these associations because retinal light exposures at night can suppress the hormone melatonin and/or disrupt circadian entrainment to the local 24-h light-dark cycle. The present study examined whether it was valid to use satellite photometry and self-reports of brightness to characterize light, as it might stimulate the circadian system and thereby affect BC incidence. Calibrated photometric measurements were made at the bedroom windows and in the bedrooms of a sample of female school teachers, who worked regular dayshifts and lived in a variety of satellite-measured sky brightness categories. The light levels at both locations were usually very low and were independent of the amount of satellite-measured light. Calibrated photometric measurements were also obtained at the corneas of these female school teachers together with calibrated accelerometer measurements for seven consecutive days and evenings. Based upon these personal light exposure and activity measurements, the female teachers who participated in this study did not have disrupted light-dark cycles like those associated with rotating shiftworkers who do exhibit a higher risk for BC. Rather, this sample of female school teachers had 24-h light-dark and activity-rest patterns very much like those experienced by dayshift nurses examined in an earlier study who are not at an elevated risk of BC. No relationship was found between the amount of satellite-measured light levels and the 24-h light-dark patterns these women experienced. It was concluded from the present study that satellite photometry is unrelated to personal light exposures as they might affect melatonin suppression and/or circadian disruption. More generally, photometric devices calibrated in terms of the operational characteristics of the human circadian system must be used to meaningfully link LAN and BC incidence. (Authors correspondence: E-mail: ream@rpi.edu)

Rat photoreceptor circadian oscillator strongly relies on lighting conditions

Mammalian retina harbours a self-sustained circadian clock able to synchronize to the light : dark (LD) cycle and to drive cyclic outputs such as night-time melatonin synthesis. Clock genes are expressed in distinct parts of the tissue, and it is presently assumed that the retina contains several circadian oscillators. However, molecular organization of cell type-specific clockworks has been poorly investigated. Here, we questioned the presence of a circadian clock in rat photoreceptors by studying 24-h kinetics of clock and clock output gene expression in whole photoreceptor layers isolated by vibratome sectioning. To address the importance of light stimulation towards photoreceptor clock properties, animals were exposed to 12 : 12 h LD cycle or 36 h constant darkness. Clock, Bmal1, Per1, Per2, Cry1, Cry2, RevErbα and Rorβ clock genes were all found to be expressed in photoreceptors and to display rhythmic transcription in LD cycle. Clock genes in whole retinas, used as a reference, also showed rhythmic expression with marked similarity to the profiles in pure photoreceptors. In contrast, clock gene oscillations were no longer detectable in photoreceptor layers after 36 h darkness, with the exception of Cry2 and Rorβ. Importantly, transcripts from two well-characterized clock output genes, Aanat (arylalkylamine N-acetyltransferase) and c-fos, retained sustained rhythmicity. We conclude that rat photoreceptors contain the core machinery of a circadian oscillator likely to be operative and to drive rhythmic outputs under exposure to a 24-h LD cycle. Constant darkness dramatically alters the photoreceptor clockwork and circadian functions might then rely on inputs from extra-photoreceptor oscillators.

Circadian rhythm and suprachiasmatic nucleus alterations in the mouse model of mucopolysaccharidosis IIIB

Mucopolysaccharidosis IIIB (MPSIIIB) is a lysosomal storage disease characterised by progressive central nervous system degeneration in patients, with death usually in the late teens. Serious behavioural problems have been reported in children at the early stages of the disease, such as hyperactivity and severe sleep disturbances, which suggest alterations in circadian rhythms. We investigated the circadian rhythm of locomotor activity of young and old MPSIIIB mice, under a 24-h light–dark (LD) cycle and under constant darkness (DD), and also examined neuropeptide expression in the suprachiasmatic nucleus (SCN), site of the principal biological pacemaker. We show that MPSIIIB mice have higher activity levels during the light (resting) phase of the LD cycle, together with weaker circadian rhythms, and a longer active phase due to a late peak of activity, in both LD and DD. In addition, young MPSIIIB mice showed shorter phase delays in response to a light pulse in DD. Increased lysosomal storage, neuroinflammation and changes in the expression of Arginine Vasopressin and Vasointestinal Polypeptide, two circadian neuropeptides, were observed in the SCN, which may be in part responsible for the changes in circadian behaviour observed in MPSIIIB mice. These findings suggest an alteration of the circadian system in MPSIIIB mice, and may inform better clinical management of circadian, sleep and behavioural disturbances in patients with MPSIII.

Photoperiodism of diapause induction in Thyrassia penangae (Lepidoptera: Zygaenidae)

Thyrassia penangae enters winter diapause as a prepupa in a cocoon. Photoperiodism of diapause induction was systematically investigated in this moth. The photoperiodic response curves under 24-h light–dark cycles showed that this insect is a typical long-day species. The critical daylength was 13 h 30 min at 25 °C, 13 h at 30 °C and 12 h 20 min at 28 °C. Transferring experiments from a short day (LD 12:12) to a long day (LD 15:9) or vice versa indicated that photoperiodic sensitivity mainly occurs during the larval period. In experiments using non-24-h light–dark cycles, when the length of photophase exceeded the critical daylength (13.5 h), was diapause inhibited effectively, even when the length of scotophase exceeded the critical nightlength (10.5 h). Only when a long scotophase was combined with a short photophase, diapause was induced effectively. This result suggests that daylength measurement is more important than nightlength measurement in T. penangae. Night interruption experiments under 24-h light–dark cycles exhibited two points of apparent light sensitivity, but the photosensitive position was highly influenced by temperature and the length of scotophase. Nanda–Hamner experiments failed to reveal the involvement of a circadian system in this photoperiodic time measurement. All light–dark cycles from LD 12:12 to LD 12:72 resulted in a short day response, and all cycles from LD 14:4 to LD 14:72 resulted in a long day response, suggesting that photoperiodic time measurement in this moth is performed by a day-interval timer or an hourglass-like clock.

Diapause induction and clock mechanism in the cabbage butterfly Pieris melete Ménétriés

Photoperiodic control of diapause induction was systematically investigated in the cabbage butterfly, Pieris melete, which enters summer and winter diapause as a pupa. Summer and winter diapause are induced principally by short and long scotophases, respectively; the intermediate scotophases (11–12 h) permit pupae to develop without diapause. Photoperiodic responses under 24-h light–dark cycles at 16.9, 18, 20 and 22 °C showed that the hibernation response was temperature compensated, whereas aestivation response was strongly temperature-dependent. The incidence of diapause for both aestivation and hibernation showed a decline at the ultra-short and ultra-long scotophases. Experiments using non-24-h light–dark cycles showed that the length of the scotophase played an essential role in the determination of diapause. The highest photosensitivity differed under hibernation and aestivation conditions. With a 3 × LD 12:12 interruption, a maximal inhibition of aestivation occurred in the L3/2 stage, and of hibernation it occurred in the L4/0 stage. A long-night of LD 10:14 induced hibernation diapause but inhibited aestivation diapause and, conversely, a short-night of LD 14:10 inhibited hibernation diapause but induced aestivation diapause. With a 1-h light pulse at LD 11:13, a maximal inhibition of hibernation occurred 3 h before lights-on (late scotophase), whereas, with a 1-h light pulse at LD 12.5:11.5, a maximal induction of aestivation occurred 2–3 h after the onset of darkness (early scotophase). Nanda-Hamner and Bünsow experiments failed to reveal the involvement of a circadian system, suggesting that the photoperiodic time measurement for diapause induction in this butterfly resembles an hourglass-like timer or a damped circadian oscillator.

Amplitude of circadian oscillations entrained by 24-h light–dark cycles

An intriguing property of circadian clocks is that their free-running period is not exactly 24 h. Using models for circadian rhythms in Neurospora and Drosophila, we determine how the entrainment of these rhythms is affected by the free-running period and by the amplitude of the external light–dark cycle. We first consider the model for Neurospora, in which light acts by inducing the expression of a clock gene. We show that the amplitude of the oscillations of the clock protein entrained by light–dark cycles is maximized when the free-running period is smaller than 24 h. Moreover, if the amplitude of the light–dark cycle is very strong, complex oscillations occur when the free-running period is close to 24 h. In the model for circadian rhythms in Drosophila, light acts by enhancing the degradation of a clock protein. We show that while the amplitude of circadian oscillations entrained by light–dark cycles is also maximized if the free-running period is smaller than 24 h, the range of entrainment is centered around 24 h in this model. We discuss the physiological relevance of these results in regard to the setting of the free-running period of the circadian clock.