Natural Variation in the Drosophila melanogaster Clock Gene Period Modulates Splicing of Its 3′-Terminal Intron and Mid-Day Siesta

Drosophila melanogaster exhibits circadian (≅24 hr) regulated morning and evening bouts of activity that are separated by a mid-day siesta. Increases in daily ambient temperature are accompanied by a progressively longer mid-day siesta and delayed evening activity. Presumably, this behavioral plasticity reflects an adaptive response that endows D. melanogaster with the ability to temporally optimize daily activity levels over a wide range of physiologically relevant temperatures. For example, the shift in activity towards the cooler nighttime hours on hot days might minimize the risks associated with exposure to mid-day heat, whereas on cold days activity is favored during the warmer daytime hours. These temperature-induced shifts in the distribution of daily activity are partly based on the thermal sensitive splicing of an intron found in the 3′ untranslated region (UTR) of the circadian clock gene termed period (per). As temperature decreases, splicing of this 3′-terminal intron (termed dmpi8) is gradually increased, which is causally linked to a shorter mid-day siesta. Herein we identify several natural polymorphisms in the per 3′ UTR from wild-caught populations of flies originating along the east coast of the United States. Two non-intronic closely spaced single nucleotide polymorphisms (SNPs) modulate dmpi8 splicing efficiency, with the least efficiently spliced version associated with a longer mid-day siesta, especially at lower temperatures. Although these SNPs modulate the splicing efficiency of dmpi8 they have little to no effect on its thermal responsiveness, consistent with the notion that the suboptimal 5′ and 3′ splice sites of the dmpi8 intron are the primary cis-acting elements mediating temperature regulation. Our results demonstrate that natural variations in the per gene can modulate the splicing efficiency of the dmpi8 intron and the daily distribution of activity, providing natural examples for the involvement of dmpi8 splicing in the thermal adaptation of behavioral programs in D. melanogaster.     

How a circadian clock adapts to seasonal decreases in temperature and day length

We show that a thermosensitive splicing event in the 3' untranslated region of the mRNA from the period (per) gene plays an important role in how a circadian clock in Drosophila adapts to seasonally cold days (low temperatures and short day lengths). The enhanced splicing of this intron at low temperatures advances the steady state phases of the per mRNA and protein cycles, events that significantly contribute to the preferential daytime activity of flies on cold days. Because the accumulation of PER is also dependent on the photosensitive TIMELESS (TIM) protein, long photoperiods partially counteract the cold-induced advances in the oscillatory mechanism by delaying the daily increases in the levels of TIM. Our findings also indicate that there is a temperature-dependent switch in the molecular logic governing cycles in per mRNA levels.     

Circadian clock of Drosophila montana is adapted to high variation in summer day lengths and temperatures prevailing at high latitudes

Photoperiodic regulation of the circadian rhythms in insect locomotor activity has been studied in several species, but seasonal entrainment of these rhythms is still poorly understood. We have traced the entrainment of activity rhythm of northern Drosophila montana flies in a climate chamber mimicking the photoperiods and day and night temperatures that the flies encounter in northern Finland during the summer. The experiment was started by transferring freshly emerged females into the chamber in early and late summer conditions to obtain both non-diapausing and diapausing females for the studies. The locomotor activity of the females and daily changes in the expression levels of two core circadian clock genes, timeless and period, in their heads were measured at different times of summer. The study revealed several features in fly rhythmicity that are likely to help the flies to cope with high variation in the day length and temperature typical to northern summers. First, both the non-diapausing and the diapausing females showed evening activity, which decreased towards the short day length as observed in the autumn in nature. Second, timeless and period genes showed concordant daily oscillations and seasonal shifts in their expression level in both types of females. Contrary to Drosophila melanogaster, oscillation profiles of these genes were similar to each other in all conditions, including the extremely long days in early summer and the cool temperatures in late summer, and their peak expression levels were not locked to lights-off transition in any photoperiod. Third, the diapausing females were less active than the non-diapausing ones, in spite of their younger age. Overall, the study showed that D. montana clock functions well under long day conditions, and that both the photoperiod and the daily temperature cycles are important zeitgebers for seasonal changes in the circadian rhythm of this species.     

Clock gene daily profiles and their phase relationship in the rat suprachiasmatic nucleus are affected by photoperiod

Rhythmicity of the rat suprachiasmatic nucleus (SCN), a site of the circadian pacemaker, is affected by daylength; that is, by the photoperiod. Whereas various markers of rhythmicity have been followed, so far there have been no studies on the effect of the photoperiod on the expression of the clock genes in the rat SCN. To fill the gap and to better understand the photoperiodic modulation of the SCN state, rats were maintained either under a long photoperiod with 16 h of light and 8 h of darkness per day (LD16:8) or under a short LD8:16 photoperiod, and daily profiles of Per1, Cry1, Bmal1 and Clock mRNA in darkness were assessed by in situ hybridization method. The photoperiod affected phase, waveform, and amplitude of the rhythmic gene expression as well as phase relationship between their profiles. Under the long period, the interval of elevated Per1 mRNA lasted for a longer and that of elevated Bmal1 mRNA for a shorter time than under the short photoperiod. Under both photoperiods, the morning and the daytime Per1 and Cry1 mRNA rise as well as the morning Bmal1 mRNA decline were closely linked to the morning light onset. Amplitude of Per1, Cry1, and Bmal1 mRNA rhythms was larger under the short than under the long photoperiod. Also, under the short photoperiod, the daily Clock mRNA profile exhibited a significant rhythm. Altogether, the data indicate that the whole complex molecular clockwork in the rat SCN is photoperiod dependent and hence may differ according to the season of the year.     

Effect of temperature on circadian rhythm controlling the crepuscular activity of the burying beetle Nicrophorus quadripunctatus Kraatz (Coleoptera: Silphidae)

Locomotor activity rhythm was examined at various temperatures under a 16 h light : 8 h dark photoperiod (LD 16:8) or LD 12:12 using adults of the burying beetle Nicrophorus quadripunctatus. At 20°C, the locomotor activity of the beetles showed a bimodal daily pattern with two peaks around lights on and lights off under both photoperiods. This bimodal activity rhythm persisted under constant darkness; therefore, the activity of adult N. quadripunctatus is controlled by a circadian clock. Adults showed a bimodal activity pattern for temperatures ranging from 15 to 25°C. The evening peak of the daily activity was earlier at lower temperatures. These findings suggest that in the field, N. quadripunctatus shows crepuscular activity, and is active earlier in the afternoon in cooler seasons. In this species, therefore, temperature appears to play an important role in the determination of daily activity patterns.     

Low temperature limits photoperiod control of smolting in atlantic salmon through endocrine mechanisms

We have examined the interaction of photoperiod and temperature in regulating the parr-smolt transformation and its endocrine control. Atlantic salmon juveniles were reared at a constant temperature of 10 degrees C or ambient temperature (2 degrees C from January to April followed by seasonal increase) under simulated natural day length. At 10 degrees C, an increase in day length [16 h of light and 8 h of darkness (LD 16:8)] in February accelerated increases in gill Na(+)-K(+)-ATPase activity, whereas fish at ambient temperature did not respond to increased day length. Increases in gill Na(+)-K(+)-ATPase activity under both photoperiods occurred later at ambient temperature than at 10 degrees C. Plasma growth hormone (GH), insulin-like growth factor, and thyroxine increased within 7 days of increased day length at 10 degrees C and remained elevated for 5-9 wk; the same photoperiod treatment at 2 degrees C resulted in much smaller increases of shorter duration. Plasma cortisol increased transiently 3 and 5 wk after LD 16:8 at 10 degrees C and ambient temperature, respectively. Plasma thyroxine was consistently higher at ambient temperature than at 10 degrees C. Plasma triiodothyronine was initially higher at 10 degrees C than at ambient temperature, and there was no response to LD 16:8 under either temperature regimen. There was a strong correlation between gill Na(+)-K(+)-ATPase activity and plasma GH; correlations were weaker with other hormones. The results provide evidence that low temperature limits the physiological response to increased day length and that GH, insulin-like growth factor I, cortisol, and thyroid hormones mediate the environmental control of the parr-smolt transformation.     

Temporal pattern in foraging behaviour of the Indian pond heron,Ardeola grayii at Ratanpur,Chhattisgarh, India

In avian population, the biological clock is synchronized with the photoperiod as a significant time cue. However, information on feeding behaviour of the Indian Pond Heron, Ardeola grayii at Ratanpur, Chhattisgarh, India is not available. In present study we examined the effect of ‘time of the day’ and ‘photoperiod’ on daytime feeding behaviour of A. grayii at Ratanpur (85º17’E longitude and 22º3’N latitude), Chhattisgarh, India. The different feeding techniques of A. grayii, were recorded for two consecutive days each during long days (May and June 2014) and during short days (December 2014 and January 2015). One-way ANOVA (using SPSS 16.0) was applied to find out the effects of ‘photoperiod’ and ‘time of the day’ on daytime feeding activity with respect to frequency of feeding techniques. The rhythms in daytime feeding activity were evaluated using the Cosinor rhythmometry at 24 h and 12 h. The present study revealed that the A. grayii adopted three major techniques viz., slowly walking, probing and striking on which the slowly walking and striking was popularly used feeding technique as compared to the probing technique during the feeding. Moreover, the A. grayii is found to be bimodal viz., morning and evening type during both of long and short days. Furthermore, bird was more active during short day as compared to the long day.     

Cleavage site of a major yolk protein (MYP) determined by cDNA isolation and amino acid sequencing in sea urchin,Hemicentrotus pulcherrimus

The grey mouse lemur (Microcebus murinus) is a small nocturnal primate exhibiting daily torpor. In constant ambient temperature (22-24 degrees C), body temperature (Tb) and locomotor activity were monitored by telemetry in animals exposed to short (SP: 10 h light/day) or long (LP: 14 light/day) photoperiods. They were first fed ad libitum for 8 days and then subjected to 80% restricted feeding for 8 more days. During ad libitum feeding, locomotor activity was significantly lower in SP-exposed animals than in LP-exposed animals. Whatever the photoperiod, animals entered daily hypothermia within the first hours following the light onset. Depth of daily hypothermia increased irregularly under SP exposure, whereas minimal daily Tb was constantly above 35 degrees C under LP exposure. After the transfer from long photoperiod to short photoperiod corresponding to the induction of seasonal fattening, locomotor activity and depth of controlled daily hypothermia did not change significantly. In contrast, food restriction led to a significant increase in locomotor activity and in frequency of daily torpor (Tb<33 degrees C) and body temperature reached minimum values averaging 25 degrees C. However, SP-exposed animals exhibited lower minimal daily Tb and higher torpor duration than LP exposed animals. Therefore, daily torpor appears as a rapid response to food restriction occurring whatever the photoperiod, although enhanced by short photoperiod.     

How photoperiod influences body temperature selection in Lacerta viridis

Summary European green lizards, Lacerta viridis, show a distinct annual cycle in their day and nighttime selected body temperature (T _b) levels when monitored under natural photoperiod. The amplitude between daily photophase and scotophase temperatures varies throughout the year. Highest body temperatures with smallest day/night variation are selected from May through July. Throughout fall, the difference between day and nighttime selected T _b levels increases. Lizards inevitably enter a state of winter dormancy which terminates daily rhythmicity patterns. Under natural photoperiodic conditions, cessation of dormancy occurs spontaneously by mid-March, regardless whether high temperatures are available or not. Lacerta viridis respond to an artificial long photoperiod (16 h light, 8 h dark) at all times of the year with modifications in both diel patterns and levels of selected T _b to summer-like conditions. When, however, the natural photoperiod at different phases in the annual cycle is held constant for six to eight weeks, T _b selection of Lacerta viridis also remains stable at the level corresponding to the prevailing photoperiod. These results implicate that the photoperiod is a more prominent Zeitgeber for seasonal cueing of temperature selection than has been surmised in the past. Further, we suggest that the large variations recorded in daily T _b cycles do not imply that this lizard is an imprecise thermoregulator, but rather indicates an important integral process necessary for seasonal acclimatization.     

Photoperiodic regulation of PER1 and PER2 protein expression in rat peripheral tissues

Circadian oscillations in biological variables in mammals are controlled by a central pacemaker in the suprachiasmatic nuclei (SCN) of the hypothalamus which coordinates circadian oscillators in peripheral tissues. The molecular clockwork responsible for this rhythmicity consists of several clock genes and their corresponding proteins that compose interactive feedback loops. In the SCN, two of the genes, Per1 and Per2, show circadian rhythmicity in their expression and protein production. This SCN rhythmicity is modified by the length of daylight, i.e. the photoperiod. The aim of the present study was to find out whether profiles of PER1 and PER2 proteins in peripheral organs are also affected by the photoperiod. Rats were maintained under a long photoperiod with 16 h of light and 8 h of darkness per day (LD 16:8) and under a short, LD 8:16, photoperiod. The PER1 and PER2 daily profiles were measured in peripheral organs by Western blotting. The photoperiod affected significantly the PER1 profile in livers and the PER2 profile in lungs and hearts. In lungs, PER2 in the cytoplasmic, but not in the nuclear fraction, was affected significantly. The effect of the photoperiod on PER1 profiles in peripheral organs appears to differ from that in the SCN.     

Novel masking effects of light are revealed in Drosophila by skeleton photoperiods

Here, we show that in a skeleton photoperiod where all midday light is removed from a standard laboratory 12:12 LD photoperiod, a large diurnal peak of activity is revealed that is continuous with the E peak seen in constant dark (DD). We further show that the circadian clock gene tim regulates light-dependent masking of daytime activity, but the clock gene per does not. Finally, relative to wild-type flies, mutants for both of these clock genes showed increased nighttime activity in the skeleton photoperiod but not in the standard photoperiod. This result suggests that nighttime activity is suppressed by the intact circadian clock, and in its absence, by exposure to a standard photoperiod. These results support and extend the literature addressing the complex interactions between masking and clock-controlled components of overt circadian rhythms.     

Long and short isoforms of Neurospora clock protein FRQ support temperature-compensated circadian rhythms

The large (l) and small (s) isoforms of FREQUENCY (FRQ) are elements of interconnected feedback loops of the Neurospora circadian clock. The expression ratio of l-FRQ vs. s-FRQ is regulated by thermosensitive splicing of an intron containing the initiation codon for l-FRQ. We show that this splicing is dependent on light and temperature and displays a circadian rhythm. Strains expressing only l-FRQ or s-FRQ support short and long temperature-compensated circadian rhythms, respectively. The thermosensitive expression ratio of FRQ isoforms influences period length in wt. Our data indicate that differential expression of FRQ isoforms is not required for temperature compensation but rather provides a means to fine-tune period length in response to ambient temperature.     

Photoperiod, reproduction, and immunity in select strains of inbred mice

The purpose of this study was to determine whether decreased day lengths affect reproduction or the immune system in inbred mice. Irrespective of a nocturnal pineal melatonin rise, the signal for day length information, body and testis weights were the same in various strains 8 weeks after transfer from long to short days (16 to 8 h of light/day) compared to mice that remained in long days. Serum testosterone was unaffected by the photoperiod shift. The second goal was to determine whether the shift from long to short days influenced lymphocyte populations in spleen or blood, as well as innate and cell-mediated immune cell functions in C3H/HeN mice, an inbred strain with a robust melatonin rhythm. By flow cytometry, a stable percentage and number of B cells, T cells, and natural killer cells were identified in spleen from mice in both long and short days during the day and night. This complement of immunophenotypes in spleen suggests that equivalent functional capabilities persist in secondary lymphoid tissue of mice irrespective of day length. This was supported by findings that cytolytic activity by splenic natural killer cells (innate immunity) and antigen-induced T cell-dependent B cell antibody production (adaptive immunity) were similar in mice in long and short days. In blood, cell numbers but not helper T cell subset percentages (i.e., naive, memory, cytotoxic, or activated) were augmented in mice in short compared to long days, a consequence of increased circulating B cells. Day length differences in certain immunophenotypes in circulation may forecast photoperiod-mediated alterations in responsiveness to pathogens that are associated with a change in season. At night, the reduced proportion of cytotoxic T cells (long and short days), as well as increases in the percentage of activated T cells (long days), B cells (short days), and NK cell activity (long and short days) relative to daytime, suggests that surveillance and function by select immunophenotypes may adapt to circadian transitions even in highly inbred species. Thus, inbred mice retain capabilities for photoperiod to influence trait-specific aspects of immune cell but not reproductive function.