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.     

亚洲玉米螟两个不同地理种群对暗期干扰的滞育反应

为了探明亚洲玉米螟Ostrinia furnacalis (Guenée)不同地理种群对暗期干扰的滞育反应, 本文在研究了亚洲玉米螟南昌种群（28°41′N, 115°53′E）和哈尔滨种群（44°56′N, 127°10′E）光周期反应的基础上, 在室内分别测试了25℃和28℃温度下1 h的光脉冲干扰光周期L9∶D15和L12∶D12对这两个不同地理种群幼虫滞育抑制的影响。结果表明： 在25℃和28℃下, 哈尔滨种群的临界日长分别比南昌种群延长了1 h 40 min和2 h; 在所有测试的暗期干扰实验中, 除了极少数光脉冲干扰点外, 南昌种群幼虫滞育的发生率显著低于哈尔滨种群; 1 h光脉冲干扰光周期L9∶D15的滞育发生率明显低于干扰光周期L12∶D12, 且前者表现了暗期的中间对光脉冲最敏感, 而后者显示了暗期的初期对光脉冲最敏感; 28℃下光脉冲对滞育的抑制效果强于25℃。这些结果进一步提示, 即使在同种昆虫中, 如果地理种群和实验条件不同, 暗期干扰对滞育抑制的效果也可能不同。     

Photoperiodic induction of pupal diapause in the flesh fly,Sarcophaga crassipalpis: embryonic sensitivity

Summary The last two days of embryonic development are crucial in programming pupal diapause in the flesh fly,Sarcophaga crassipalpis. Short daylength (greater than 10 1/2h of darkness) during this interval permits expression of diapause while long daylength during this brief sensitive stage eliminates the potential for diapause. Length of scotophase rather than photophase programs the diapause although three hours of light is needed to separate tandem dark periods. Early in the scotophase, photosensitivity is restricted to blue light (less than 540 nm). The scotophase can be divided into 4 phases according to the effect of light breaks on diapause expression. During Phase I (0–6 h after scotophase onset) embryos are highly sensitive to light interruption and diapause is effectively eliminated. A period of insensitivity to light, Phase II, extends from 6–hh after onset of scotophase. Light breaks at 10–11h coincide with the critical scotophase length and result in a partial reduction of diapause. In Phase IV, the scotophase reaction is complete and diapause competence is preserved even in the presence of light. Although light breaks result in elimination of diapause throughout Phase I, recovery time from a 1 h light break (length of darkness needed to counter the effect of a light break) differs dramatically depending upon when the light break is presented. Early in Phase I (0–3h) recovery from light interruption is rapid, while late in Phase I (4–6h), the effects of light are not readily reversible. The scotophase reaction thus appears to follow a step-wise progression rather than represent a simple linear response. We present a molecular model that could account for the dynamics of the scotophase reaction.     

Diapause induction, maintenance and termination in the rice stem borer Chilo suppressalis (Walker)

The rice stem borer, Chilo suppressalis, enters facultative diapause as fully grown larvae in response to short-day conditions during the autumn. Our results showed that the critical night length for diapause induction in C. suppressalis was between 10 h 22 min and 10 h 45 min at 22, 25 and 28 °C, 11 h 18 min at 31 °C, and between 10 h 5 min and 10 h 20 min under field conditions (average temperature ranged from 27.2 to 30.7 °C). The diapause incidence declined in ultra-long nights (18-22 h scotophases) and DD, and increased in ultra-short nights (2-6 h scotophases) and LL. Moreover, we found that the third instar was the stage most sensitive to the photoperiod, and night length played an essential role in the initiation of diapause. Night-interruption experiments with a 1-h light pulse at LD 12:12 (light 12:dark 12) exhibited two troughs of diapause inhibition, with one occurring in early scotophase and the other in late scotophase. Field observations for six years showed that most larvae entered winter diapause in August in response to declining day lengths, despite the high temperatures prevailing during August. By periodically transferring the field-collected overwintering larvae to different photoperiods and temperatures, the results showed that photoperiod had a significant influence on diapause development during the early phase of diapause, while high temperature significantly accelerated the termination of larval diapause.     

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.     

Interacting effects of photophase and scotophase on the diapause response of the Indian meal moth, Plodia interpunctella

The diapause-programming response to photoperiod in Plodia interpunctella was analyzed by exposing larvae to various 24-h and non-24-h regimes of light and darkness. The response to 24-h regimes indicated three photoperiodic parameters—a critical scotophase, a minimal photophase, and a minimal scotophase for a full expression of the response. The critical response was based on dark-time measurement, because disruption of the scotophase abolished the response and the diapause incidence varied as a function of scotophase in non-24-h regimes. The critical scotophase varied with the duration of the preceding photophase. Prevention of diapause by single or double-night interruptions of long scotophases could be explained by resetting of the dark-time measurement. The effect of a light pulse was modified by the quantitative interaction of light and dark reactions. The sensitivity to resetting by a light pulse seemed to be decreased in the early scotophase with an increasing duration of the preceding light period. Therefore, the significance of light in the photoperiodic response was something more than delimiting scotophase for the time measurement.     

Inheritance of the photoperiodic response controlling imaginal summer diapause in the cabbage beetle, Colaphellus bowringi

Adults of the cabbage beetle Colaphellus bowringi display a summer diapause in response to the exposure of their larvae to long photoperiods. In the present study, the inheritance of the photoperiodic response controlling summer diapause in C. bowringi by crossing a high diapause strain (D strain) with a laboratory selected nondiapause strain (N strain) was investigated under different photoperiods at 22, 25 and 28 °C. The beetles in both reciprocal crosses and backcrosses showed a clear short-day response for the induction of diapause at all temperatures, similar to that of the D strain, suggesting that photoperiodic response of this beetle is heritable. The diapause incidences in the progeny from all the crosses under LD 15:9 or LD 12:12 at 25 °C suggest that genetic and genetic-environmental interactions are involved in diapause induction. The incidence of diapauses in F₁ progeny was significantly lower than that in the D × D strain but significantly higher than that in the N × N strain, indicating that the diapause capability is inherited in an incomplete dominant manner. The incidence of diapause was greater among the offspring of hybrid females when those females had a D strain mother or grandmother rather than a N strain mother or grandmother, indicating that maternal effects on diapause induction are stronger than paternal effects. The laboratory selected nondiapause strain also showed a short-day photoperiodic response at a low temperature of 22 °C, indicating that the photoperiodic photoreceptor and photoperiodic clock still function in the nondiapause strain.     

Some effects of low temperature on the photoperiodic clock in Drosophila triauraria

Abstract. Prior studies revealed that the photoperiodic clock in Drosophila triauraria operates during the scotophase and the processes measuring scotophase are composed of two phases: the A phase which lasts 6–7 h from the onset of the scotophase and the B phase which follows the A phase. In this study the photoperiodic clock in this species was analysed by low-temperature pulses. Low temperature (5C) or a low-temperature (2C) pulse of 3 or 4 h decreased the induction of diapause when applied during the scotophase, but not when applied within the photophase. The low-temperature pulse showed two maxima of the effect, 0–4 h and 8–10 h after the onset of the scotophase, approximately corresponding to the A and B phases, respectively. It is considered that low temperature retards the time measurement processes operating during these phases. In the period which was rather insensitive to the diapause-preventing action of low temperature, i.e. 5-7h after the onset of the scotophase, substances or stimuli accumulated during the A phase may be converted to information required for operation of the machinery in the B phase. Experiments using a combination of low-temperature and light pulses suggested that low-temperature decreases the sensitivity to light, especially in the B phase.     

An “hourglass-like” feature in the photoperiodic time measurement in Diatraea grandiosella (Pyralidae)

Photoperiodic time measurement of Diatraea grandiosella, a Pyralid moth, was investigated for its role in the determination of diapause by using various night-interruption protocols. The photoperiodic-response curve showed a temperature dependence under short days, whereas at long days it was stable in the range between 20 and 30°C. A light pulse averted diapause most effectively when it was placed 6 h after lights-off. Earlier pulses were less effective but gradually increased in effectiveness as the time of the pulse approached the critical time, i.e. 6 h after lights-off. A strong conversion in response appeared as the pulse passed the critical time. The same response pattern was observed both in diapause induction and termination. The species required a long light pulse to avert diapause completely, even if the light pulse was placed during the critical time; 45 min was required to reverse diapause in 50% of the individuals. The most crucial event for photoperiodic time measurement in this species was whether a night phase 6–7.5 h after lights-off was illuminated or not. This hourglass-like feature was also shown in more complex night-interruption protocols with 2–3 light pulses. A possible relation of this phenomenon to the cricadian pacemaker was sought. The Dual System Theory failed to account for most of the features for the photoperiodic time measurements of D. grandiosella found in this study.     

Diapause induction and photoperiodic clock in Chilo suppressalis (Walker) (Lepidoptera: Crambidae)

The mature larvae of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Crambidae) enters facultative diapause in response to short‐day conditions in the autumn (August–September). Diapause induction and photoperiodic clock mechanism were investigated in C. suppressalis larvae reared on an artificial diet in the present study. The critical night length for diapause induction was about 9 h 53 min to 10 h 39 min at 22 to 28°C. The third‐instar larvae were found to be relatively sensitive to diapause induction. Photoperiodic response under non‐24‐h light–dark cycles showed that scotophase length played an essential role in the induction of larval diapause in C. suppressalis, and consecutive exposure to long‐night cycles was necessary for a high diapause incidence. In the Nanda–Hamner experiment, diapause incidence peaked at scotophase of 12 h and dropped rapidly at scotophases > 24 h. In the Bünsow experiment, diapause incidence was clearly suppressed, especially at the light pulse located 8 h in the scotophase. Both the Nanda–Hamner and Bünsow experiments showed no rhythmic fluctuations with a period of about 24 h; thus the photoperiodic clock in C. suppressalis is a non‐oscillatory hourglass timer or a rapidly damping circadian oscillator.     

Photoperiodic counter of diapause induction in Pseudopidorus fasciata (Lepidoptera: Zygaenidae)

Induction of larval diapause is a photoperiodically controlled event in the life history of the moth Pseudopidorus fasciata. In the present study, the photoperiodic counter of diapause induction has been systematically investigated. The required day number (RDN) for a 50% response was determined by transferring from a short night (LD 16:8) to a long night (LD 12:12) or vice versa at different times after hatching, The RND differed significantly between short- and long-night cycles at different temperatures. The RDN for long-night cycles at 20, 22, 25 and 28 degrees C was 11.5, 9.5, 7.5 and 8.5 days, respectively. The RDN for short-night cycles was 3 days at 22 degrees C and 5 days at 20 degrees C indicating that the effect of one short night was equivalent to the effect of 2-3 long nights effect. Night-interruption experiments of 24h photoperiods by a 1 h light pulse showed that the most crucial event for the photoperiodic time measurement in this moth was whether the length of pre-interruption (D(1)) or the post-interruption (D(2)) scotophases exceeded the critical night length (10.5 h). If D(1) or D(2) exceeded 10.5 h diapause was induced. The diapause-averting effect of a single short-night cycle (LD 16:8) against a background of long nights (LD 12:12) showed that the photoperiodic sensitivity was greatest during the first 7 days of the larval period and the highest sensitivity was on the fourth day. Both non-24 and 24 h light-dark cycle experiments revealed that the photoperiodic counter in P. fasciata is able to accumulate both long and short nights during the photosensitive period, but in different ways. The information from short-night cycles seems to be accumulated one by one in contrast to long-night cycles where six successive cycles were necessary for about 50% diapause induction and eight cycles for about 90% diapause. These results suggest the accumulation of long-night and short-night cycles may be based on different mechanisms.     

Effects of thermoperiod on photoperiodic determination of larval diapause in Ostrinia nubilalis

Some effects of thermoperiods on the photoperiodic determination of larval diapause in the European corn borer, Ostrinia nubilalis are reported. Thermoperiods and photoperiods were shown to interact to a highly significant degree in the induction of diapause. Diapause determination was found to be sensitive to the duration and temperature of the cryoscotophase of the thermophotoperiod; the thermophotophase characteristics exerted only minor influence. Cryoscotophase temperatures of 10°C or lower caused an unexpected increase in the apparent critical nightlength for diapause determination. A 0°C cryoscotophase effectively prevented the photoperiodic determination of diapause. Experimental investigation of the effects of 0°C cryoscotophases produced evidence that such low-temperature cryoscotophases may suppress the biological clock functions regulating the determination of diapause. Photoperiodic regimes in which 0°C pulses were inserted at successive points in 24-h cycle evoked responses suggesting that the early scotophase (saturation phase) was more strongly influenced by the low temperature pulse than was the later scotophase (scotonon); low temperature had little effect on the light-dependent processes (photonon) associated with the photophase.     

Diapause Induction and Termination in Hyphantria cunea (Drury) (Lepidoptera: Arctiinae)

The fall webworm, Hyphantria cunea (Drury), enters facultative diapause as a pupa in response to short-day conditions during autumn. Photoperiodic response curves showed that the critical day length for diapause induction was 14 h 30 min, 14 h 25 min and 13 h 30 min at 22, 25 and 28°C, respectively. The photoperiodic responses under non-24 h light–dark cycles demonstrated that night length played an essential role in the determination of diapause. Experiments using a short day length interrupted by a 1-h light pulse exhibited two troughs of diapause inhibition and the effect of diapause inhibition was greater in the early scotophase than in the late scotophase. The diapause-inducing short day lengths of 8, 10 and 12 h evoked greater intensities of diapause than did 13 and 14 h. Diapause can be terminated without exposure to chilling, but chilling at 5°C for 90 and 120 d significantly accelerated diapause development, reduced mortality, and synchronized adult emergence. Additionally, the potential for H. cunea from the temperate region (Qingdao) to emerge and overwinter under field conditions in subtropical regions (Nanchang) of China was evaluated. Pupae that were transferred to Nanchang in early July showed a 60% survival rate and extremely dispersed pupal period (from 12 to 82 days), suggesting that some pupae may undergo summer diapause. Diapausing temperate region pupae that were moved out-of-doors in Nanchang during October showed approximately 20% overwintering survival; moreover, those pupae that overwintered successfully emerged the next spring during a period when their host plants would be available. The results indicate that this moth has the potential to expand its range into subtropical regions of China.     

暗期干扰对棉铃虫两个不同地理种群滞育抑制作用的比较

至今,所测试昆虫的光周期反应均表明,光周期反应对暗期干扰高度敏感,短暂的光脉冲都可在不同程度上逆转长夜效应,抑制滞育的发生。在研究了棉铃虫Helicoverpa armigera泰安种群(36.15°N,116.59°E)和喀佐种群(41.34°N,120.27°E)光周期反应的基础上,在滞育诱导的短光周期下(L12:D12和L9:D15),分别测试了暗期不同时段1h光脉冲对这两个不同地理种群滞育抑制的影响。25和22℃下的光周期反应显示了泰安种群在长暗期11—14 h的滞育率均显著低于喀佐种群;泰安种群的临界暗长分别为11.7 h和11.5 h,喀佐种群分别为10.5 h和10.3 h,泰安种群均比喀佐种群长1.2 h。在所测试的暗期干扰实验中,除了极少数光脉冲干扰点外,泰安种群蛹滞育率显著低于喀佐种群,但两者的滞育反应曲线基本相似。在短光周期L9:D15下,泰安种群和喀佐种群均显示了光脉冲落入暗期的第9—11小时最有效地抑制了滞育的发生。在短光周期L12∶D12下,泰安种群和喀佐种群在25℃时均显示了光脉冲落入暗期的第3—4小时和第10小时导致了最低的滞育发生;但在22℃时,喀佐种群只在暗期的第3—4小时显示了最高的滞育抑制。这些结果揭示了偏南的泰安种群对暗期干扰的敏感性强于偏北的喀佐种群,但这两个地理种群的最高光敏感位点基本相同。     

Photoperiodic control of adult diapause in the predacious mite, Amblyseius potentillae: repeated diapause induction and termination

ABSTRACT. Photoperiodic control of facultative reproductive diapause was examined in females of the phytoseiid mite, Amblyseius potentillae (Garman). Full diapause was induced by rearing the mites from egg to adult under short-day photoperiods. Egg-laying females, however, which had experienced a long-day photoperiodic regime during pre-imaginal development, could still be induced to enter diapause when they were transferred to a short-day regime. Diapause development proceeded slowly under a short-day photoperiod, but was accelerated under long days. During diapause development under short days the photoperiodic sensitivity of the females diminished gradually and ultimately disappeared completely. However, after completion of diapause, sensitivity to photo-period reappeared. A second diapause could be induced in post-diapause females under short-day photoperiods and completed again under long-day photoperiods. These results show that A. potentillae remains sensitive to diapause-inducing and diapause-averting daylengths during the adult stage and that a second diapause may be induced after completion of the first one.     

Diapause induction and clock mechanism in the cabbage beetle, Colaphellus bowringi (Coleoptera: Chrysomelidae)

Photoperiodic control of diapause induction was investigated in the short-day species, Colaphellus bowringi, which enters summer and winter diapause as adult in the soil. Photoperiodic responses at 25 and 28 degrees C revealed a critical night length between 10 and 12 h; night lengths > or =12 h prevented diapause, whereas night lengths <12 h induced summer diapause in different degree. Experiments using non-24-h light-dark cycles showed that the duration of scotophase played an essential role in the determination of diapause. Night-interruption experiments with T=24 h showed that diapause was effectively induced by a 2-h light pulse in most scotophases; whereas day-interruption experiments by a 2-h dark break had a little effect on the incidence of diapause. The experiments of alternating short-night cycles (LD 16:8) and long-night cycles (LD 12:12) during the sensitive larval period showed that the information of short nights as well as long nights could be accumulated. Nanda-Hamner experiments showed three declining peaks of diapause at 24 h circadian intervals. Bünsow experiments showed two very weak peaks for diapause induction, one being 8 h after lights-off, and another 8 h before lights-on, but it did not show peaks of diapause at a 24 h interval. These results suggest that the circadian oscillatory system constitutes a part of the photoperiodic clock of this beetle but plays a limited role in its photoperiodic time measurement.     

The effect of larval temperature and photoperiod on the incidence of larval diapause in the blowfly, Calliphora vicina

ABSTRACT. The blowfly, Calliphora vicina Meigen (Diptera: Calliphoridae), displays a maternally induced larval diapause. Progeny of adults exposed to short days enter diapause if the larval temperature is 15C or below; exposing adults to long days, or larvae to temperatures above 15C, results in non-diapause. By keeping progeny from short-day parents at the parental temperature of 23.5C for various lengths of time before transfer to 11C, it could be shown that the diapause-averting effect of high temperature operates in the late wandering stage of larva, possibly at the moment of the decision for the brain-ring gland complex to release PTTH/ecdysone at the diapause/non-diapause stage.
Although photoperiodic induction in the blowfly is maternal, previous work had shown that the larvae were also slightly sensitive to photoperiod. From the present study it is evident that also the eggs are photo-sensitive.     

Effects of photoperiod and temperature on pupal diapause induction of grape berry moth Lobesia botrana

Abstract. The grape berry moth, Lobesia botrana Denn. & Sciff. (Lepidoptera: Tortricidae), one of the most injurious pest of grape berries in Greece, is a multivoltine species that overwinters as diapausing pupae. The effects of several diel and non-diel photoperiods and of temperature, experienced by eggs and larvae, on pupal diapause induction were investigated. The diapause response curve was of Type I (long day type) and the determining factor was the duration of scotophase (> 11 h), regardless of the duration of photophase. However, at very short (< 4 h) photoperiods, the incidence of diapause was also high. Diapause was positively and significantly correlated with the egg-larval developmental time, pupal mortality and the duration of the pupal stage. Eggs and larvae reared under LD 12 : 12 h photoperiod and various temperatures (from 12 to 30 °C) produced diapausing pupae (almost 100%), but the duration of the pupal stage (intensity of diapause) increased with increasing temperature. Under continuous darkness, however, the percentage diapause decreased with increasing temperature. Single and double 1-h light pulses were applied systematically at various times during the scotophase of six diapause-inducing diel photoperiods. Two photosensitive points in time (called A and B) were revealed, during which illumination resulted in a significant decrease of diapause induction. The decrease was much greater during the first sensitive period (early in scotophase) rather than in the second (late in the scotophase).     

The role of the main photophase on dark-time measurement used for diapause determination in the Indian meal moth, Plodia interpunctella

Abstract.  The Indian meal moth Plodia interpunctella Hubner (Lepidoptera: Pyralidae) measures night length and enters diapause as a last-instar larva. To examine the role of photophase on dark-time measurement, the main LD 7 : 17 h photoperiod is disrupted by various lengths of darkness at 25 °C. When the light phase is not disrupted, the incidence of diapause is 76%. As the dark pulse disrupting a 7-h photophase becomes longer, the incidence of diapause decreases. To detect the dynamic kinetics of the time-measuring process, the main scotophase of 17 h is scanned by a 2-h light pulse. When the dark pulse in a 7-h photophase is fixed at 1 h after dawn and its duration is varied systematically from 1 to 3 h, or when the end of the dark pulse is fixed at 1 h before dusk, diapause is prevented completely by a 2-h light pulse inserted in the middle of 17-h darkness. These results are compared with those of a single night interruption of a 17-h scotophase with a 2-h light pulse but with an intact 7-h photophase. The disruption of a 7-h photophase by a dark pulse shifts the descending and ascending slopes of the response curve to some extent toward dawn and dusk, respectively, indicating that the dark pulse tends to shorten the critical length of dark time for diapause induction. When the main photophase (7 h) is interrupted by a 1-h dark pulse at 3–4 h after dawn, the 2-h scanning light pulse in the main scotophase (17 h) appears to act effectively as a dusk signal in the early scotophase. However, those in the mid- and late scotophase do not define the critical night length from dusk as sharply as for the critical night length from a 2-h light pulse to dawn. The results indicate the importance of photophase in the dark-time measurement.