Behavioral and molecular analysis of laboratory populations of drosophila melanogaster selected for early and late adult emergence

Abstract

Circadian clocks enhance the chances of survival of organisms living under periodic environment by enabling them to efficiently anticipate periodic events in their environment, because precisely and appropriately timed behavioral and metabolic processes are thought to confer greater adaptive advantage than randomly occurring activities. Among clock regulated phenomena in insects adult emergence (eclosion) rhythm is one of the most extensively studied and perhaps the best understood after activity/rest cycle. Although each individual emerges as an adult only once in its life cycle, gating of this event is under the control of an on-going oscillation present during development (Saunders, 1992). Consequently, certain intervals of time in a day constitute the “forbidden zone of eclosion”, whereas a brief period of time during which adults emerge forms the “allowed zone” (also referred to as the “gate” of eclosion) (Pittendrigh, 1954). The gating is often so stringent that even if developing flies are mature enough to emerge but fail to do so during the gate they remain within the puparium until the next gate opens (Saunders, 1992). My thesis is motivated by the need for an unequivocal, systematic and rigorous approach for studying the evolution of circadian waveform of adult emergence in fruit flies Drosophila melanogaster. The main aim of my thesis is to study the effect of selection on timing of adult emergence on circadian clocks, clockrelated rhythm and life history traits. For this purpose I derived four genetically independent, random mating, large populations each of early and late populations of D. melanogaster by selecting for individuals that emerge during “lights-on” (morning hours) and “lights-off” (evening hours) under 12:12 hr LD cycles. I assessed the direct response to selection by comparing the number of flies that emerged out of the morning and v evening windows of selection in the selected and control populations at regular intervals of 10-15 generations to trace the evolutionary trajectory of changes over 55 generations of selection. I observed that D. melanogaster populations respond to selection on timing of adult emergence, since the percentage of flies that emerge out of the morning window after 55 generations of selection is about 60% in the early populations while it is reduced to almost one third (~33%) in the late populations, and remain unchanged at ~45% in the controls. The percentage of flies that emerged out of the evening window in the late populations (~24%) is about thrice as much as in the early populations (~8%), while it remain unchanged at ~16% in the controls. To investigate the consequence of selection on circadian clocks we assayed eclosion and activity rhythms in the selected and control populations under LD and constant dark (DD) conditions. Under 12:12 hr LD cycles, the primary peaks of eclosion in the early populations are taller and occur earlier than the controls, while those in the late populations are relatively flatter and occur later than the controls. The early flies start and end activity earlier and are generally more active during the morning hours, while the late flies start and end activity later and are more active during the evening hours. In order to test the robustness of the circadian phenotypes of the selected populations we assayed the eclosion and activity rhythms under short (8:16 hr), normal (12:12 hr) and long (16:8 hr) photoperiodic regimes at the 70th generation. Although the overall eclosion and activity patterns of flies are influenced by the photoperiodic conditions, the relative phase separations between the selected and control populations are maintained; the time course and waveform of the early populations remain phase advanced relative to the controls, while those of the late populations are phase delayed relative to the controls. Consistent with the rhythmic expression of the vi selected populations under LD cycles, even the circadian periodicity of eclosion and activity rhythms under DD condition is significantly longer in the late populations compared to the early populations. Such alterations in circadian phenotypes, borne out of heritable changes in genetic architecture in response to imposed selection pressure (and not because of random genetic drift or some unknown environmental or non-genetic effect) suggest that the time course and waveform of adult emergence and activity rhythms in D. melanogaster evolve as correlated responses to selection on the timing of adult emergence. We further investigated the consequence of selection on life history traits such as pre-adult developmental time and adult lifespan in unmated (virgin) flies from the selected and control populations. The development time of the selected populations was altered as compared to the controls; the early populations develop faster than the controls under LD as well as DD conditions whereas the late flies take longer to develop under both the regimes. This suggests that selection for early and late adult emergence causes correlated change in the duration of pre-adult development. The lifespan of virgin flies from the selected populations depends upon the timing of their emergence. For example, the morning emerging early flies live longer than those that emerged during the evening, while the evening emerging late flies live longer than those that emerged during the morning. This, to the best of my knowledge is the first study of its kind demonstrating that morning emergence is adaptive for early populations while evening emergence is adaptive for the late populations. In the last part of my thesis I have described studies on the molecular clocks of the early, control and late populations. I have assayed the levels of transcripts of clock vii genes period and clock (per and clk) in the selected and control populations. Consistent with the circadian phenotypes, the peak of per mRNA in the late populations is phase delayed by about 4 hr compared to the early and control populations. Similarly, the peak of the clk mRNA in the late populations occurs about 4 hr later compared to the early and control populations. Besides the transcript levels even the ratio of spliced to unspliced variants in the per 3’ UTR is significantly altered among the selected populations; the early populations display an overall suppressed spliced form of per throughout the day with a peak at ZT14 (2 hr after lights-off), whereas the late populations have generally higher levels of spliced form of per with a peak at ~ZT20. In other words, the spliced form of per peaks early in the early populations and later in the late flies. These findings are consistent with earlier studies that implicated role of per splicing in the determination of the phase of evening activity peak (Collins et al., 2004; Majercak et al., 2004). Although the time course and waveform of the early and control populations appear to have diverged from each other and from the late populations, they do not differ from each other with regards to their free running period (τ), which raises a conundrum as to how clocks with similar τ could result in different circadian phenotypes under LD cycles. This is possible only if their clocks are differentially sensitive to light stimuli. Indeed the results of my experiment reveal that, the early populations undergo greater phase advance in their eclosion and activity rhythms in response to brief light stimuli administered during the late subjective night (Circadian time 20, CT20) and a modest delay during early part of the subjective night (CT14), whereas the late populations display exactly opposite response at these two phases. Given that circadian clocks underlying eclosion and locomotor activity in the selected and control populations viii respond differently to brief light stimuli presented at CT14 and CT20, we decided to estimate the TIM levels at CT14 and CT20 to investigate the state of the molecular clock of the selected populations following a brief exposure to light. The late flies show drastic reduction in TIM levels at CT14 associated with large phase delays, while the early flies show lesser reduction in TIM levels associated with smaller delays, whereas at CT20, the light-dependent degradation is found to be greater in the early populations compared to the controls. Interestingly, the late populations show similar reduction to those of the early populations at this phase suggesting to a specific role for photoreceptor molecule (CRY) in the late populations. However, the role of CRY is yet to be ascertained in our selection lines. The results of the experiments described in my thesis suggest that fruit flies D. melanogaster evolve morning and evening preference for adult emergence and activity as a result of periodic selection pressure imposed on the timing of adult emergence. As a consequence the early populations develop faster than the late populations and morning emergence becomes adaptive for the early populations and evening emergence for late populations. These studies also underscore the significance of timing of rhythmic phenomena for organisms living under periodic environments, and suggest a possible mechanism by which circadian rhythms may have evolved and/or fine-tuned by periodic biotic and abiotic factors of nature.