BACKGROUND
most holometabolous insects including fruit flies d. melanogaster go through three discrete developmental stages namely egg, larvae and pupae.
at an ambient temperature of 25°c, eggs typically take 18–24 h to hatch followed by the larval stage which spans for ~4 days, during which developing larvae pass through three instars  <cit> .
the pupal stage starts after the third instar larval stage, lasts for another 4 days, subsequently leading to wing-pigmentation followed by adult emergence, hence the entire pre-adult developmental duration of drosophila spans ~9 days.
wing-pigmentation is considered to be the last stage of the fly development and therefore, no further major change in pupa is expected to take place thereafter  <cit> .
circadian clocks have been implicated in the temporal regulation of pre-adult development in d. melanogaster <cit>  as several studies have reported that the pre-adult development time and clock period show positive correlation  <cit> .
furthermore, studies on insects including drosophila have reported rhythmicity in several developmental events such as egg-hatching  <cit> , pupation  <cit>  and wing-pigmentation  <cit> , implying a role of circadian clocks in timing pre-adult stages.
while adult emergence in insects including drosophila is under the control of circadian clocks  <cit> , evidence suggests that clocks begin ticking in the fly as early as the third instar larval stage and is functional for the most part of pre-adult development  <cit> .
additionally, drosophila larvae are known to show rhythmicity in light avoidance behaviour  <cit> , which is probably the earliest and the only clock-driven pre-adult behaviour reported thus far in drosophila.
light/dark  cycles are known to be one of the strongest zeitgebers for the adult emergence rhythm of fruit flies and is known to play a key role in entraining  the circadian clocks present during early developmental stages  <cit> .
adult emergence rhythm in drosophila is known to be entrained by a wide range of ld cycles  <cit> , causing a significant impact on its pre-adult developmental duration; speeding it up under constant light , relative to 12:12 h ld cycles and slowing it down under dd  <cit> .
in addition, pre-adult development time in drosophila is reported to be positively correlated with the period of ld cycles , suggesting a role of period of circadian rhythms and/or ld cycles in the regulation of pre-adult developmental duration  <cit> .
while it is known that the duration of pre-adult development would be greatly affected by environmental ld cycles  <cit> , which of the developmental stages would be affected the most is still unknown.
previously we had reported a corollary to the above - that populations of d. melanogaster subjected to selection for faster pre-adult development under dd, evolve shorter development time  and circadian clocks with free-running period ~ <dig>  h shorter than the controls  <cit> .
additionally, we had reported that speeding up of development in these populations is achieved by concurrent reduction in the duration of almost all pre-adult stages  <cit> .
evolution of faster running circadian clocks in the faster developing  flies suggests a link between circadian clocks and development time, similar to what has been implied in several previous studies  <cit> .
considering the ability of circadian clocks to entrain to a wide range of ld cycles, and a sizable difference in development time between the faster developing and control populations  <cit> , in the present study, we decided to examine the effects of interaction between circadian clocks and light regimes in the timing of pre-adult developmental stages by assaying the duration of several pre-adult stages in the selected faster developing  and control baseline developing  populations under three light/dark  cycles  and two constant conditions .
RESULTS
egg-hatching time assay
since egg-hatching in drosophila typically lasts for  <dig> to 24 h and it would require a very high resolution in data to pick-up any difference between the selected and control flies, we estimated this duration only in ll, t <dig> and dd conditions.
under all the three light regimes, the egg-hatching waveform of the fd flies was shifted earlier compared to the bd controls , and the average duration of the egg-stage was shorter in the fd flies compared to the bd controls by ~ <dig>  h under ll, ~ <dig>  h in t <dig> and ~ <dig>  h in dd .
anova revealed a statistically significant effect of light regime , stock ; however, the effect of l × s interaction was statistically not significant .
under all the three light regimes, egg-hatching time of the fd flies was significantly shorter than the bd controls .
however, the difference in the egg-hatching time between the fd and bd flies did not differ statistically between the three environmental conditions .
shortening of egg-hatching duration in the fd flies, under all the three light regimes, indicates that response to selection for faster development overrides the effects of light regimes .
df
df
f
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egg-hatching time assay under ll, t <dig> and dd
pupation-time assay under ll, t <dig>  t <dig>  t <dig> and dd
wing-pigmentation time assay under ll, t <dig>  t <dig>  t <dig> and dd
egg-to-adult development time assay under ll, t <dig>  t <dig>  t <dig> and dd
df
df
f
p
egg-hatching time difference
pupation-time difference
wing-pigmentation time difference
egg-to-adult development time difference
pupation and wing-pigmentation time assays
these assays were performed under five different regimes .
under all the five assay light regimes, pupation  and wing-pigmentation time  of the fd flies was significantly shorter than the bd controls.
the pupation-time difference between the fd and bd flies under ll, t <dig>  t <dig>  t <dig> and dd conditions was about  <dig>   <dig> ,  <dig>   <dig> and  <dig>  h respectively .
under t <dig>  the fd flies pupated mostly in the dark and the bd flies in the light, while in t <dig>  flies from both the stocks pupated during the light phase.
however, under t <dig>  pupation of the fd flies spanned over both dark and light phases, while that of the bd flies was mostly restricted to the dark phase and partly to the light phase.
the fd flies pupated earlier than the bd controls under both ll and dd .
anova on the pupation-time data revealed a statistically significant effect of l, s and l × s interaction .
post-hoc multiple comparisons using tukey’s test revealed that under all the five light regimes, pupation-time of the fd flies was significantly shorter compared to the bd controls.
pupation-time of both the stocks was shorter under ll followed by t <dig> and t <dig>  while it was longer in t <dig> and dd .
anova on the pupation-time difference  data revealed a statistically significant effect of l  with the difference being smaller under ll, t <dig>  t <dig> and t <dig>  compared to dd.
thus, under entraining as well as rhythm-abolishing conditions, clock-mediated difference in pupation-time between the fd and bd flies is significantly reduced, while under free-running condition, the difference persisted.
these results suggest that circadian clocks regulate pupation-time in drosophila.
the difference in wing-pigmentation time between the fd and bd stocks was about 14 h in ll, 11 h in t <dig>  12 h in t <dig>   <dig>  h in t <dig> and 18 h in dd .
under t <dig>  wing-pigmentation of the fd flies started in the light phase and was completed in the dark phase, while in the bd flies it started in the dark and ended during the light phase.
in t <dig>  wing-pigmentation of the fd flies was confined to the light phase, while that of bd flies was confined to the dark phase .
under t <dig>  pigmentation occurred during both light and dark phases in the fd flies, while in the bd flies it occurred only during the dark phase, and under both ll or dd, the fd flies pigmented earlier than the bd controls .
anova on the wing-pigmentation time data revealed a statistically significant effect of l, s and l × s interaction .
post-hoc multiple comparisons using tukey’s test revealed that under all five light regimes, wing-pigmentation time of the fd flies was significantly shorter than the bd controls, and the mean wing-pigmentation time of both the populations was shortest under ll, followed by t <dig> and t <dig>  while it was longest in t <dig> and dd .
the wing-pigmentation time difference between the two stocks was significantly shorter under ll, t <dig>  t <dig> and t <dig> compared to dd .
anova on the difference data revealed a statistically significant effect of l , indicating that under ll and entrained conditions , clock-mediated difference in pigmentation-time between the fd and bd flies is significantly reduced while it persists in free-running condition .
this suggests that circadian clocks regulate timing of wing-pigmentation in drosophila.
egg-to-adult development time
after  <dig> generations of selection, the difference in pre-adult development time between the fd and bd stocks was about 15 h in ll, 15 h in t <dig>  13 h in t <dig>  15 h in t <dig> and 18 h in dd .
pre-adult emergence profiles of the fd and bd flies showed similar pattern across all the light regimes, with the fd flies emerging consistently earlier than the bd controls .
however, difference in development time between the two populations under ll and three ld cycles was significantly smaller than that in dd .
under t <dig>  both fd and bd flies emerged in the dark and emergence ended in the light phase of the next cycle, and in t <dig>  emergence of the fd flies was confined to the light phase, while that of the bd flies to the dark phase .
under t <dig>  the fd flies started emerging in the middle of the light phase, continued emerging in the dark phase and their emergence was completed only in the light phase of the next cycle, while that of the bd flies was confined only to the light phase .
anova revealed a statistically significant effect of l, s and l × s interaction .
post-hoc multiple comparisons using tukey’s test revealed that under all the five light regimes, egg-to-adult development time of the fd flies was significantly shorter than the bd controls, and the mean development time of both the populations was shortest under ll followed by t <dig> and t <dig>  and was longest under t <dig> and dd .
anova on the difference data revealed that the effect of l was statistically significant .
the difference in development time  between the selected and control flies under the entraining conditions  and rhythm abolishing condition  was significantly reduced compared to that under free-running condition .
these results suggest that circadian clocks regulate the duration of pre-adult development in d. melanogaster.
free-running period of adult emergence and activity/rest rhythms
we estimated the period of adult emergence and activity/rest rhythms from the emergence and activity data collected after  <dig> generations of selection .
the fd populations had a significantly shorter  period of adult emergence rhythm  compared to the bd controls .
similarly, the fd populations had a significantly shorter  period of activity/rest rhythm  compared to the bd controls .
these results suggest that selection for faster pre-adult development results in a correlated shortening of clock period in the fd populations compared to the controls.
discussion
our previous study had shown that, under dd, developmental events such as egg-hatching, pupation and wing-pigmentation occur earlier in the fd flies than in the bd controls  <cit> .
we hypothesized that if the duration of pre-adult stages is entirely clock-mediated, the difference in the duration between the fd and bd flies would disappear  under the three ld cycles, due to the fact that circadian clocks of flies from both the stocks would run at the same pace owing to circadian entrainment, and  under ll, wherein their clocks would be rendered dysfunctional.
indeed, the results of our study revealed that the difference in the duration of pre-adult stages between the fd and bd stocks was significantly reduced under the three ld cycles and ll compared to dd.
however, exposure to ld or ll regimes did not eliminate the difference in the duration of pre-adult developmental stages between the fd and bd flies completely, which suggests that circadian clocks only partly regulate the timing of pre-adult developmental stages in d. melanogaster.
exposure to light  significantly reduces the egg-hatching time but has no effect on the difference between the fd and bd stocks, suggesting that egg-hatching is light-mediated but a clock-independent process.
in the current study we have estimated the time of egg-hatching with reference to the egg-stage , which is likely to represent the total duration of egg-stage.
our results are also consistent with the findings of previous studies which reported that a single light pulse administered immediately after the egg-hatching stage is sufficient to entrain the circadian clocks of drosophila while the clocks start ticking only at the third instar stage  <cit> .
earlier studies have shown that pupation is gated in mosquitoes such as anopheles gambiae <cit>  and aedes taeniorhynchus <cit> , and the period  mrna expression during pupation in fruit flies d. melanogaster is under circadian clock control  <cit> .
moreover, several developmental events such as egg-hatching  <cit> , pupation  <cit>  and wing-pigmentation  <cit> , have been reported to show rhythmicity, which implies that circadian clocks are likely to interact with several gated events during the egg-hatching, pupation and wing-pigmentation stages.
such gated events and the transition time from one stage to another is likely to create constraints on the developmental rates, which in turn would cause reduction in the proportionate differences in the duration of pupal , wing-pigmentation  and pre-adult developmental stages of the fd and bd flies .
concurrently, difference in the duration of the two pre-adult stages between the fd and bd stocks was found to be greater under dd compared to the three ld or ll conditions .
this could partly be due to the fact that the difference in clock period between the fd and bd stocks persists under dd, while it disappears under ld and ll conditions.
under all the five light regimes, pupation, wing-pigmentation and adult emergence in the control populations began only after these processes were nearly completed in the selected populations .
this indicates that the impact of selection for faster pre-adult development is much stronger than that of light-regimes; however, difference in the duration of pre-adult stages between the fd and bd flies varied between the entraining, rhythm abolishing and free-running light regimes .
this suggests that time-to-pupation, wing-pigmentation and adult emergence is a function of the period of circadian clocks, implying that interaction of light regimes and circadian clocks is a key determinant for the timing of pre-adult developmental events in drosophila.
in insects, timing of ecdysone  release is known to trigger pupal development  <cit> ; its premature release speeds-up development while delayed release slows it down  <cit> .
in hornworms manduca sexta, opening of gate during the larval stage is believed to be the signal that regulates the timing of release of prothoracicotropic hormone  <cit> .
thus, it is suggested that modulation of pre-adult development time may be due to the altered timing of prothoracicotropic hormone release which is primarily caused by the altered timing and the duration of gate opening at different developmental stages.
although in our present study we did not estimate ecdysone levels, the observation that clocks of both selected and control flies appear to have entrained to all the imposed ld cycles at egg , larval  and pupal  stages provides an indirect evidence for the difference in the timing of ecdysone release being regulated by the gating of various developmental stages.
the timing of adult emergence in drosophila depends upon a number of factors including developmental states, phase and period of circadian rhythms and on the external environmental conditions  <cit> .
ld cycles restrict emergence of adults to a narrow window of time called as “allowed zone” or “gate” of emergence  <cit> .
since circadian rhythms in drosophila are abolished under ll, gating of emergence is likely to be absent in this condition, and therefore, developing individuals would enter the subsequent developmental stages without any delay, thereby speeding up the pre-adult developmental stages  <cit> .
on the other hand under ld cycles, duration of developmental events is likely to be determined by an interaction between the developmental states and circadian gating created by the ld cycles, which is likely to be altered depending on the length of the ld cycles and the timing of light/dark phases  <cit> .
under dd, where circadian clocks free-run, development time would be determined by some interactions between the developmental states and circadian clocks, and thus development time of flies in this regimes would be comparable to that in t <dig> .
therefore, timing of pre-adult developmental events in drosophila is expected to follow the trend of being fastest under ll < t20 < t <dig> or dd < t <dig>  the results of our present study are consistent with these trends and with earlier findings as it shows that the mean development time of the two major developmental stages is shorter under ll and t <dig> and longer in t <dig>  t <dig> and dd  <cit> .
as expected, flies take similar amount of time to develop under t <dig> and dd .
interestingly, lack of emergence gating under ll does not supersede the extent of shortening of development under t <dig>  among the ld cycles, development time was shortest under t <dig>  followed by t <dig> and t <dig>  with flies taking shorter time to develop under t <dig> compared to t <dig>  this hints at the possibility of a threshold, beyond which the pre-adult development of drosophila cannot be slowed down any further, at least not by light, thus constraining the nature of correlation between development time and the period of light regime beyond a particular limit.
interestingly, the timing of developmental events in the selected as well as control populations which accompanied small but statistically significant difference in clock period  was considerably affected by light regimes; the difference in the duration of developmental stages is shortened under ll and the three ld cycles compared to dd.
however, irrespective of the state of temporal organization, flies selected to develop faster as pre-adults had shorter egg-to-adult developmental duration compared to the controls, under the three ld cycles and ll conditions.
this suggests that to a large extent, the difference in development time between the selected and control flies is independent of the difference in their clock period.
therefore, development time differences between the two stocks under different light regimes alone cannot be taken as evidence to suggest the role of circadian clocks in the regulation of the timing of pre-adult developmental stages in drosophila.
since clock independent and light mediated pathways are also likely to play some role in the regulation of development time, genetic experiments involving flies with modified circadian clocks would be helpful.
however, mutant lines are often inbred, which may yield spurious genetic correlations between fitness components  <cit> .
therefore, in such studies, use of mutant flies may not be an ideal choice  <cit> , and the best strategy would be to examine such correlations in natural populations, where sufficient variation in clock period and development time is likely to exists.
the other alternative would be to examine such correlations in large replicate populations, selected for different clock period values.
several studies have shown that most insect species are sensitive to light especially in the blue-green region of spectra , while a few species show sensitivity up to the red end of the spectrum  <cit> .
moreover, a study in which eggs of pink bollworm pectinophora gossypielia were exposed to monochromatic light after the midpoint of embryogenesis, showed initiation of the egg-hatching rhythm optimally between  <dig> and 480 nm and a sharp cut-off above 520 nm  <cit> .
similarly, larvae of the cabbage white butterfly pieris brassicae were found to be more sensitive to wavelengths between  <dig> to 520 nm while insensitive to red light  <cit> .
in a previous study, it was reported that drosophila larvae are highly sensitive to 500 nm , 420 nm  and 380 nm   <cit> .
since the spectral peaks of the light source used in our study correspond to 570 nm  and 420 nm , it is likely that the light input pathways affecting the duration of pre-adult stages comprise of photopigments sensitive to  <dig> and 420 nm .
moreover, two additional peaks in the spectrum corresponding to 570–590 nm , 620 nm  suggests the likelihood of these wavelengths also influencing the developmental rates of fruit flies.
the faster developing  populations have evolved small but consistent  and reproducibly  shorter free-running period of circadian adult emergence and activity/rest rhythm  compared to the controls .
however, it is arguable whether such changes are really significant because it is easy to obtain a small period change without substantial impact on the core clock mechanism.
there are numerous examples in circadian biology literature where differences in behavioural rhythms are not found to be correlated with the molecular or neural mechanisms.
for instance, differences in the activity/rest rhythm seen in fruit fly populations maintained under semi-natural conditions did not correlate with changes at the molecular or neural levels  <cit> .
similarly difference in activity/rest rhythm of different drosophila species was found to occur in spite of very little difference at the molecular and neural levels  <cit> .
the fact that all the four populations of the faster developing flies underwent changes in the same direction , suggests that the period differences are not because of random genetic drift but due to the imposed selection for faster pre-adult development.
multigenic traits such as clock period showing a consistent and reproducible change is a testament to adaptive evolution of circadian clocks as a correlated response to selection for faster development.
furthermore, it is unlikely that the genetic architecture underlying the circadian phenotype would permit large changes in circadian period because of multigenic control.
our results are also consistent with the notion that while direct response to selection on a trait is limited to few times  the standard deviation of the mean trait value, correlated responses are much smaller .
in summary, across successive stages of development, circadian clocks of drosophila under various light regimes interact with several “gated” developmental events.
provided development is a light regime-mediated clock-controlled event, stage-specific clock or light regime-dependent effects can only be observed by a manipulation in the clock speed which can then be detected in terms of relative difference in development time across the light regimes.
our study reveals that unlike the egg stage, most pre-adult developmental stages in fruit flies d. melanogaster are light sensitive and clock-controlled.
CONCLUSIONS
we found that several pre-adult developmental stages of d. melanogaster are susceptible to light and its duration is determined by the interaction between developmental clocks and circadian gating created by ld cycles, suggesting that interaction of light regimes and circadian clocks modulate the timing of pre-adult events in drosophila.

 most holometabolous insects including fruit flies d. melanogaster go through three discrete developmental stages namely egg, larvae and pupae. at an ambient temperature of 25°c, eggs typically take 18–24 h to hatch followed by the larval stage which spans for ~4 days, during which developing larvae pass through three instars. the pupal stage starts after the third instar larval stage, lasts for another 4 days, subsequently leading to wing-pigmentation followed by adult emergence, hence the entire pre-adult developmental duration of drosophila spans ~9 days. wing-pigmentation is considered to be the last stage of the fly development and therefore, no further major change in pupa is expected to take place thereafter. circadian clocks have been implicated in the temporal regulation of pre-adult development in d. melanogaster as several studies have reported that the pre-adult development time and clock period show positive correlation. furthermore, studies on insects including drosophila have reported rhythmicity in several developmental events such as egg-hatching, pupation and wing-pigmentation, implying a role of circadian clocks in timing pre-adult stages. while adult emergence in insects including drosophila is under the control of circadian clocks, evidence suggests that clocks begin ticking in the fly as early as the third instar larval stage and is functional for the most part of pre-adult development. additionally, drosophila larvae are known to show rhythmicity in light avoidance behaviour, which is probably the earliest and the only clock-driven pre-adult behaviour reported thus far in drosophila. light/dark cycles are known to be one of the strongest zeitgebers for the adult emergence rhythm of fruit flies and is known to play a key role in entraining the circadian clocks present during early developmental stages. adult emergence rhythm in drosophila is known to be entrained by a wide range of ld cycles, causing a significant impact on its pre-adult developmental duration; speeding it up under constant light, relative to 12:12 h ld cycles and slowing it down under dd. in addition, pre-adult development time in drosophila is reported to be positively correlated with the period of ld cycles, suggesting a role of period of circadian rhythms and/or ld cycles in the regulation of pre-adult developmental duration. while it is known that the duration of pre-adult development would be greatly affected by environmental ld cycles, which of the developmental stages would be affected the most is still unknown. previously we had reported a corollary to the above - that populations of d. melanogaster subjected to selection for faster pre-adult development under dd, evolve shorter development time and circadian clocks with free-running period ~  h shorter than the controls. additionally, we had reported that speeding up of development in these populations is achieved by concurrent reduction in the duration of almost all pre-adult stages. evolution of faster running circadian clocks in the faster developing flies suggests a link between circadian clocks and development time, similar to what has been implied in several previous studies. considering the ability of circadian clocks to entrain to a wide range of ld cycles, and a sizable difference in development time between the faster developing and control populations, in the present study, we decided to examine the effects of interaction between circadian clocks and light regimes in the timing of pre-adult developmental stages by assaying the duration of several pre-adult stages in the selected faster developing and control baseline developing populations under three light/dark cycles and two constant conditions.  egg-hatching time assay since egg-hatching in drosophila typically lasts for to 24 h and it would require a very high resolution in data to pick-up any difference between the selected and control flies, we estimated this duration only in ll, t and dd conditions. under all the three light regimes, the egg-hatching waveform of the fd flies was shifted earlier compared to the bd controls, and the average duration of the egg-stage was shorter in the fd flies compared to the bd controls by ~  h under ll, ~  h in t and ~  h in dd. anova revealed a statistically significant effect of light regime, stock; however, the effect of l × s interaction was statistically not significant. under all the three light regimes, egg-hatching time of the fd flies was significantly shorter than the bd controls. however, the difference in the egg-hatching time between the fd and bd flies did not differ statistically between the three environmental conditions. shortening of egg-hatching duration in the fd flies, under all the three light regimes, indicates that response to selection for faster development overrides the effects of light regimes. df df f p egg-hatching time assay under ll, t and dd pupation-time assay under ll, t t t and dd wing-pigmentation time assay under ll, t t t and dd egg-to-adult development time assay under ll, t t t and dd df df f p egg-hatching time difference pupation-time difference wing-pigmentation time difference egg-to-adult development time difference pupation and wing-pigmentation time assays these assays were performed under five different regimes. under all the five assay light regimes, pupation and wing-pigmentation time of the fd flies was significantly shorter than the bd controls. the pupation-time difference between the fd and bd flies under ll, t t t and dd conditions was about, and  h respectively. under t the fd flies pupated mostly in the dark and the bd flies in the light, while in t flies from both the stocks pupated during the light phase. however, under t pupation of the fd flies spanned over both dark and light phases, while that of the bd flies was mostly restricted to the dark phase and partly to the light phase. the fd flies pupated earlier than the bd controls under both ll and dd. anova on the pupation-time data revealed a statistically significant effect of l, s and l × s interaction. post-hoc multiple comparisons using tukey’s test revealed that under all the five light regimes, pupation-time of the fd flies was significantly shorter compared to the bd controls. pupation-time of both the stocks was shorter under ll followed by t and t while it was longer in t and dd. anova on the pupation-time difference data revealed a statistically significant effect of l with the difference being smaller under ll, t t and t compared to dd. thus, under entraining as well as rhythm-abolishing conditions, clock-mediated difference in pupation-time between the fd and bd flies is significantly reduced, while under free-running condition, the difference persisted. these results suggest that circadian clocks regulate pupation-time in drosophila. the difference in wing-pigmentation time between the fd and bd stocks was about 14 h in ll, 11 h in t 12 h in t  h in t and 18 h in dd. under t wing-pigmentation of the fd flies started in the light phase and was completed in the dark phase, while in the bd flies it started in the dark and ended during the light phase. in t wing-pigmentation of the fd flies was confined to the light phase, while that of bd flies was confined to the dark phase. under t pigmentation occurred during both light and dark phases in the fd flies, while in the bd flies it occurred only during the dark phase, and under both ll or dd, the fd flies pigmented earlier than the bd controls. anova on the wing-pigmentation time data revealed a statistically significant effect of l, s and l × s interaction. post-hoc multiple comparisons using tukey’s test revealed that under all five light regimes, wing-pigmentation time of the fd flies was significantly shorter than the bd controls, and the mean wing-pigmentation time of both the populations was shortest under ll, followed by t and t while it was longest in t and dd. the wing-pigmentation time difference between the two stocks was significantly shorter under ll, t t and t compared to dd. anova on the difference data revealed a statistically significant effect of l, indicating that under ll and entrained conditions, clock-mediated difference in pigmentation-time between the fd and bd flies is significantly reduced while it persists in free-running condition. this suggests that circadian clocks regulate timing of wing-pigmentation in drosophila. egg-to-adult development time after generations of selection, the difference in pre-adult development time between the fd and bd stocks was about 15 h in ll, 15 h in t 13 h in t 15 h in t and 18 h in dd. pre-adult emergence profiles of the fd and bd flies showed similar pattern across all the light regimes, with the fd flies emerging consistently earlier than the bd controls. however, difference in development time between the two populations under ll and three ld cycles was significantly smaller than that in dd. under t both fd and bd flies emerged in the dark and emergence ended in the light phase of the next cycle, and in t emergence of the fd flies was confined to the light phase, while that of the bd flies to the dark phase. under t the fd flies started emerging in the middle of the light phase, continued emerging in the dark phase and their emergence was completed only in the light phase of the next cycle, while that of the bd flies was confined only to the light phase. anova revealed a statistically significant effect of l, s and l × s interaction. post-hoc multiple comparisons using tukey’s test revealed that under all the five light regimes, egg-to-adult development time of the fd flies was significantly shorter than the bd controls, and the mean development time of both the populations was shortest under ll followed by t and t and was longest under t and dd. anova on the difference data revealed that the effect of l was statistically significant. the difference in development time between the selected and control flies under the entraining conditions and rhythm abolishing condition was significantly reduced compared to that under free-running condition. these results suggest that circadian clocks regulate the duration of pre-adult development in d. melanogaster. free-running period of adult emergence and activity/rest rhythms we estimated the period of adult emergence and activity/rest rhythms from the emergence and activity data collected after generations of selection. the fd populations had a significantly shorter period of adult emergence rhythm compared to the bd controls. similarly, the fd populations had a significantly shorter period of activity/rest rhythm compared to the bd controls. these results suggest that selection for faster pre-adult development results in a correlated shortening of clock period in the fd populations compared to the controls. discussion our previous study had shown that, under dd, developmental events such as egg-hatching, pupation and wing-pigmentation occur earlier in the fd flies than in the bd controls. we hypothesized that if the duration of pre-adult stages is entirely clock-mediated, the difference in the duration between the fd and bd flies would disappear under the three ld cycles, due to the fact that circadian clocks of flies from both the stocks would run at the same pace owing to circadian entrainment, and under ll, wherein their clocks would be rendered dysfunctional. indeed, the results of our study revealed that the difference in the duration of pre-adult stages between the fd and bd stocks was significantly reduced under the three ld cycles and ll compared to dd. however, exposure to ld or ll regimes did not eliminate the difference in the duration of pre-adult developmental stages between the fd and bd flies completely, which suggests that circadian clocks only partly regulate the timing of pre-adult developmental stages in d. melanogaster. exposure to light significantly reduces the egg-hatching time but has no effect on the difference between the fd and bd stocks, suggesting that egg-hatching is light-mediated but a clock-independent process. in the current study we have estimated the time of egg-hatching with reference to the egg-stage, which is likely to represent the total duration of egg-stage. our results are also consistent with the findings of previous studies which reported that a single light pulse administered immediately after the egg-hatching stage is sufficient to entrain the circadian clocks of drosophila while the clocks start ticking only at the third instar stage. earlier studies have shown that pupation is gated in mosquitoes such as anopheles gambiae and aedes taeniorhynchus, and the period mrna expression during pupation in fruit flies d. melanogaster is under circadian clock control. moreover, several developmental events such as egg-hatching, pupation and wing-pigmentation, have been reported to show rhythmicity, which implies that circadian clocks are likely to interact with several gated events during the egg-hatching, pupation and wing-pigmentation stages. such gated events and the transition time from one stage to another is likely to create constraints on the developmental rates, which in turn would cause reduction in the proportionate differences in the duration of pupal, wing-pigmentation and pre-adult developmental stages of the fd and bd flies. concurrently, difference in the duration of the two pre-adult stages between the fd and bd stocks was found to be greater under dd compared to the three ld or ll conditions. this could partly be due to the fact that the difference in clock period between the fd and bd stocks persists under dd, while it disappears under ld and ll conditions. under all the five light regimes, pupation, wing-pigmentation and adult emergence in the control populations began only after these processes were nearly completed in the selected populations. this indicates that the impact of selection for faster pre-adult development is much stronger than that of light-regimes; however, difference in the duration of pre-adult stages between the fd and bd flies varied between the entraining, rhythm abolishing and free-running light regimes. this suggests that time-to-pupation, wing-pigmentation and adult emergence is a function of the period of circadian clocks, implying that interaction of light regimes and circadian clocks is a key determinant for the timing of pre-adult developmental events in drosophila. in insects, timing of ecdysone release is known to trigger pupal development; its premature release speeds-up development while delayed release slows it down. in hornworms manduca sexta, opening of gate during the larval stage is believed to be the signal that regulates the timing of release of prothoracicotropic hormone. thus, it is suggested that modulation of pre-adult development time may be due to the altered timing of prothoracicotropic hormone release which is primarily caused by the altered timing and the duration of gate opening at different developmental stages. although in our present study we did not estimate ecdysone levels, the observation that clocks of both selected and control flies appear to have entrained to all the imposed ld cycles at egg, larval and pupal stages provides an indirect evidence for the difference in the timing of ecdysone release being regulated by the gating of various developmental stages. the timing of adult emergence in drosophila depends upon a number of factors including developmental states, phase and period of circadian rhythms and on the external environmental conditions. ld cycles restrict emergence of adults to a narrow window of time called as “allowed zone” or “gate” of emergence. since circadian rhythms in drosophila are abolished under ll, gating of emergence is likely to be absent in this condition, and therefore, developing individuals would enter the subsequent developmental stages without any delay, thereby speeding up the pre-adult developmental stages. on the other hand under ld cycles, duration of developmental events is likely to be determined by an interaction between the developmental states and circadian gating created by the ld cycles, which is likely to be altered depending on the length of the ld cycles and the timing of light/dark phases. under dd, where circadian clocks free-run, development time would be determined by some interactions between the developmental states and circadian clocks, and thus development time of flies in this regimes would be comparable to that in t. therefore, timing of pre-adult developmental events in drosophila is expected to follow the trend of being fastest under ll < t20 < t or dd < t the results of our present study are consistent with these trends and with earlier findings as it shows that the mean development time of the two major developmental stages is shorter under ll and t and longer in t t and dd. as expected, flies take similar amount of time to develop under t and dd. interestingly, lack of emergence gating under ll does not supersede the extent of shortening of development under t among the ld cycles, development time was shortest under t followed by t and t with flies taking shorter time to develop under t compared to t this hints at the possibility of a threshold, beyond which the pre-adult development of drosophila cannot be slowed down any further, at least not by light, thus constraining the nature of correlation between development time and the period of light regime beyond a particular limit. interestingly, the timing of developmental events in the selected as well as control populations which accompanied small but statistically significant difference in clock period was considerably affected by light regimes; the difference in the duration of developmental stages is shortened under ll and the three ld cycles compared to dd. however, irrespective of the state of temporal organization, flies selected to develop faster as pre-adults had shorter egg-to-adult developmental duration compared to the controls, under the three ld cycles and ll conditions. this suggests that to a large extent, the difference in development time between the selected and control flies is independent of the difference in their clock period. therefore, development time differences between the two stocks under different light regimes alone cannot be taken as evidence to suggest the role of circadian clocks in the regulation of the timing of pre-adult developmental stages in drosophila. since clock independent and light mediated pathways are also likely to play some role in the regulation of development time, genetic experiments involving flies with modified circadian clocks would be helpful. however, mutant lines are often inbred, which may yield spurious genetic correlations between fitness components. therefore, in such studies, use of mutant flies may not be an ideal choice, and the best strategy would be to examine such correlations in natural populations, where sufficient variation in clock period and development time is likely to exists. the other alternative would be to examine such correlations in large replicate populations, selected for different clock period values. several studies have shown that most insect species are sensitive to light especially in the blue-green region of spectra, while a few species show sensitivity up to the red end of the spectrum. moreover, a study in which eggs of pink bollworm pectinophora gossypielia were exposed to monochromatic light after the midpoint of embryogenesis, showed initiation of the egg-hatching rhythm optimally between and 480 nm and a sharp cut-off above 520 nm. similarly, larvae of the cabbage white butterfly pieris brassicae were found to be more sensitive to wavelengths between to 520 nm while insensitive to red light. in a previous study, it was reported that drosophila larvae are highly sensitive to 500 nm, 420 nm and 380 nm. since the spectral peaks of the light source used in our study correspond to 570 nm and 420 nm, it is likely that the light input pathways affecting the duration of pre-adult stages comprise of photopigments sensitive to and 420 nm. moreover, two additional peaks in the spectrum corresponding to 570–590 nm, 620 nm suggests the likelihood of these wavelengths also influencing the developmental rates of fruit flies. the faster developing populations have evolved small but consistent and reproducibly shorter free-running period of circadian adult emergence and activity/rest rhythm compared to the controls. however, it is arguable whether such changes are really significant because it is easy to obtain a small period change without substantial impact on the core clock mechanism. there are numerous examples in circadian biology literature where differences in behavioural rhythms are not found to be correlated with the molecular or neural mechanisms. for instance, differences in the activity/rest rhythm seen in fruit fly populations maintained under semi-natural conditions did not correlate with changes at the molecular or neural levels. similarly difference in activity/rest rhythm of different drosophila species was found to occur in spite of very little difference at the molecular and neural levels. the fact that all the four populations of the faster developing flies underwent changes in the same direction, suggests that the period differences are not because of random genetic drift but due to the imposed selection for faster pre-adult development. multigenic traits such as clock period showing a consistent and reproducible change is a testament to adaptive evolution of circadian clocks as a correlated response to selection for faster development. furthermore, it is unlikely that the genetic architecture underlying the circadian phenotype would permit large changes in circadian period because of multigenic control. our results are also consistent with the notion that while direct response to selection on a trait is limited to few times the standard deviation of the mean trait value, correlated responses are much smaller. in summary, across successive stages of development, circadian clocks of drosophila under various light regimes interact with several “gated” developmental events. provided development is a light regime-mediated clock-controlled event, stage-specific clock or light regime-dependent effects can only be observed by a manipulation in the clock speed which can then be detected in terms of relative difference in development time across the light regimes. our study reveals that unlike the egg stage, most pre-adult developmental stages in fruit flies d. melanogaster are light sensitive and clock-controlled.  we found that several pre-adult developmental stages of d. melanogaster are susceptible to light and its duration is determined by the interaction between developmental clocks and circadian gating created by ld cycles, suggesting that interaction of light regimes and circadian clocks modulate the timing of pre-adult events in drosophila. 
we assayed the duration of three pre-adult stages in the faster developing and control populations under a variety of light regimes that are known to modulate circadian clocks and pre-adult development time of drosophila to examine the role of circadian clocks in the timing of pre-adult developmental stages. we reasoned that these faster developing flies could serve as a model to study stage-specific interaction of circadian clocks and developmental events with the environmental light/dark conditions. we find that the duration of pre-adult stages was shorter under constant light and short period light /dark cycles compared to the standard h day, long ld cycles and constant darkness. the difference in the duration of pre-adult stages between the fd and bd populations was significantly smaller under the three ld cycles and ll compared to dd, possibly due to the fact that clocks of both fd and bd flies are driven at the same pace in the three ld regimes owing to circadian entrainment, or are rendered dysfunctional under ll. circadian clocksfaster developmentlight regimesselectiondevelopment timepre-adult stages previously we have reported that selection for faster pre-adult development reduces development time and clock period, suggesting a role of circadian clocks in the regulation of development time in d. melanogaster. these results suggest that interaction between light regimes and circadian clocks regulate the duration of pre-adult developmental stages in fruit flies d. melanogaster. circadian clocks have been postulated to regulate development time in several species of insects including fruit flies drosophila melanogaster.
 circadian clocks have been postulated to regulate development time in several species of insects including fruit flies drosophila melanogaster. previously we have reported that selection for faster pre-adult development reduces development time and clock period, suggesting a role of circadian clocks in the regulation of development time in d. melanogaster. we reasoned that these faster developing flies could serve as a model to study stage-specific interaction of circadian clocks and developmental events with the environmental light/dark conditions. we assayed the duration of three pre-adult stages in the faster developing and control populations under a variety of light regimes that are known to modulate circadian clocks and pre-adult development time of drosophila to examine the role of circadian clocks in the timing of pre-adult developmental stages.  we find that the duration of pre-adult stages was shorter under constant light and short period light /dark cycles compared to the standard h day, long ld cycles and constant darkness. the difference in the duration of pre-adult stages between the fd and bd populations was significantly smaller under the three ld cycles and ll compared to dd, possibly due to the fact that clocks of both fd and bd flies are driven at the same pace in the three ld regimes owing to circadian entrainment, or are rendered dysfunctional under ll.  these results suggest that interaction between light regimes and circadian clocks regulate the duration of pre-adult developmental stages in fruit flies d. melanogaster. circadian clocksfaster developmentlight regimesselectiondevelopment timepre-adult stages 