Entrainment of eclosion rhythm in Drosophila melanogaster populations reared for more than 700 generations in constant light environment

In this paper, we report the results of our extensive study on eclosion rhythm of four independent populations of Drosophila melanogaster that were reared in constant light (LL) environment of the laboratory for more than 700 generations. The eclosion rhythm of these flies was assayed under LL, constant darkness (DD) and three periodic light-dark (LD) cycles (T20, T24, and T28). The percentage of vials from each population that exhibited circadian rhythm of eclosion in DD and in LL (intensity of approximately 100 lux) was about 90% and 18%, respectively. The mean free-running period (τ) of eclosion rhythm in DD was 22.85 ± 0.87 h (mean ± SD). Eclosion rhythm of these flies entrained to all the three periodic LD cycles, and the phase relationship (ψ) of the peak of eclosion with respect to “lights-on” of the LD cycle was significantly different in the three periodic light regimes (T20, T24, and T28). The results thus clearly demonstrate that these flies have preserved the ability to exhibit circadian rhythm of eclosion and the ability to entrain to a wide range of periodic LD cycles even after being in an aperiodic environment for several hundred generations. This suggests that circadian clocks may have intrinsic adaptive value accrued perhaps from coordinating internal metabolic cycles in constant conditions, and that the entrainment mechanisms of circadian clocks are possibly an integral part of the clockwork.     

Ten generations of Drosophila melanogaster reared axenically on a fatty acid-free holidic diet

Wild-type Drosophila melanogaster were axenically raised on a completely synthetic fatty acid-free diet for at least ten consecutive generations, confirming that these insects do not require dietary polyunsaturated fatty acids. Capillary column gas-chromatographic analysis of lipids extracted from adults reared on yeast medium showed a peak which cochromatographed with linoleic acid, representing about 1.2% and 0.15% of all fatty acids in phospholipids and triacylglycerols, respectively. In flies reared on the synthetic diet for one generation or for five or more generations, the linoleic acid peak was still present but in about tenfold lower proportions of total fatty acids. This was true of both phospholipid and triglyceride fractions.     

Evolution of circadian rhythms in Drosophila melanogaster populations reared in constant light and dark regimes for over 330 generations

Organisms are believed to have evolved circadian clocks as adaptations to deal with cyclic environmental changes, and therefore it has been hypothesized that evolution in constant environments would lead to regression of such clocks. However, previous studies have yielded mixed results, and evolution of circadian clocks under constant conditions has remained an unsettled topic of debate in circadian biology. In continuation of our previous studies, which reported persistence of circadian rhythms in Drosophila melanogaster populations evolving under constant light, here we intended to examine whether circadian clocks and the associated properties evolve differently under constant light and constant darkness. In this regard, we assayed activity-rest, adult emergence and oviposition rhythms of D. melanogaster populations which have been maintained for over 19 years (~330 generations) under three different light regimes – constant light (LL), light–dark cycles of 12:12 h (LD) and constant darkness (DD). We observed that while circadian rhythms in all the three behaviors persist in both LL and DD stocks with no differences in circadian period, they differed in certain aspects of the entrained rhythms when compared to controls reared in rhythmic environment (LD). Interestingly, we also observed that DD stocks have evolved significantly higher robustness or power of free-running activity-rest and adult emergence rhythms compared to LL stocks. Thus, our study, in addition to corroborating previous results of circadian clock evolution in constant light, also highlights that, contrary to the expected regression of circadian clocks, rearing in constant darkness leads to the evolution of more robust circadian clocks which may be attributed to an intrinsic adaptive advantage of circadian clocks and/or pleiotropic functions of clock genes in other traits.     

Activity rhythm of Drosophila kept in complete darkness for 1300 generations

Locomotor activity rhythms of dark stock flies of Drosophila melanogaster kept in complete darkness for 700 to 1340 generations were examined. The stock was established by the late Prof. S. Mori in November 1954 to investigate long-term effects of darkness on organisms. The activity of flies was recorded under three types of light conditions: DD after LD12:12, and DD after exposure to a 3.5 h (P3) or 7.5 h (P7) light pulse. In all of these conditions, the experimental dark flies exhibited clear circadian rhythms similar to those of control light flies. We compare our results with those of various studies on troglobites.     

Effects of extremely low‐frequency magnetic fields on the oviposition of Drosophila melanogaster over three generations

The aim of this study was to determine the effects of an extremely low‐frequency 50 Hz magnetic field (ELFMF) on the oviposition and development of Drosophila melanogaster. The results show that exposure of D. melanogaster females of generation P and all the development stages of generation F1 to an ELFMF weakened the oviposition of these insects in their subsequent generations. Bioelectromagnetics 30:687–689, 2009. © 2009 Wiley‐Liss, Inc.     

Cancer brings forward oviposition in the fly Drosophila melanogaster

Hosts often accelerate their reproductive effort in response to a parasitic infection, especially when their chances of future reproduction decrease with time from the onset of the infection. Because malignancies usually reduce survival, and hence potentially the fitness, it is expected that hosts with early cancer could have evolved to adjust their life‐history traits to maximize their immediate reproductive effort. Despite the potential importance of these plastic responses, little attention has been devoted to explore how cancers influence animal reproduction. Here, we use an experimental setup, a colony of genetically modified flies Drosophila melanogaster which develop colorectal cancer in the anterior gut, to show the role of cancer in altering life‐history traits. Specifically, we tested whether females adapt their reproductive strategy in response to harboring cancer. We found that flies with cancer reached the peak period of oviposition significantly earlier (i.e., 2 days) than healthy ones, while no difference in the length and extent of the fecundity peak was observed between the two groups of flies. Such compensatory responses to overcome the fitness‐limiting effect of cancer could explain the persistence of inherited cancer‐causing mutant alleles in the wild.     

Relationship between a metabolic rhythm and emergence rhythm in Drosophila melanogaster

Oxygen consumption and lactic acid dehydrogenase (LDH) activity were determined for Drosophila melanogaster pupae and pharate adults exposed to 12 : 12 or 1 : 23 light-dark (LD) regime. Bimodal circadian fluctuations of oxygen consumption were found in pupae and pharate adults exposed to either LD regime and organisms appeared to demonstrate an anticipatory change in oxygen consumption associated with change in illumination. The oxygen-consumption trend for the entire period spent in the puparium showed a high at the time of emergence, but the diurnal rhythm showed a low at the time of emergence suggesting that emergence occurs at a low in the diurnal cycle. Emergence maximum showed a 3 hr lead over the oxygen-consumption maximum. Changing the LD regime produced similar changes in the phasing of both oxygen consumption and emergence rhythms. LDH activity did not demonstrate a detectable circadian rhythm but did show a steady decrease during pupal and pharate adult development.     

Altitudinal variation in the circadian rhythm of oviposition in Drosophila ananassae

The effect of altitude on four basic properties of the pacemaker controlling the circadian rhythm of oviposition in two strains of Drosophila ananassae was determined. The high altitude (HA) strain from Badrinath (5123 m above sea level) had a low amplitude peak in the forenoon while the low altitude (LA) strain from Firozpur (179 m a.s.l.) had a high amplitude peak after the lights-off of LD 12:12 cycles. Free running periods in continuous darkness were about 22.6 and 27.4 h in the HA and LA strains, respectively. The light pulse phase response curve (PRC) for the HA strain showed a low amplitude and a dead zone of 8h; the ratio for the advance to delay region (A/D) was less than 1, while the PRC for the LA strain had a high amplitude, which was devoid of a dead zone and showed a ratio of A/D > 1. The magnitude of the delay phase shifts at CT 18 evoked by light pulses of 1 h duration, but varying light intensity was significantly different in the HA and LA strain, which suggests that the photic sensitivity of the clock photoreceptors mediating the phase shifts had been affected by the altitude.     

Latitude dependent arrhythmicity in the circadian oviposition rhythm of Drosophila ananassae

Latitude dependent arrhythmicity in the circadian rhythm of oviposition of Drosophila ananassae strains originating from 8.1°N to 32.7°N was studied by inbreeding them in cycles of 12 h of light at 20 lux and 12 h of darkness. The number of inbreeding generations required to initiate arrhythmicity in oviposition rhythm was dependent on the origin of latitude of the strain. The strains from the lower latitudes became arrhythmic after notably more numbers of generations than those from the higher latitudes. This might be attributed to the higher inherent degree of oviposition rhythmicity in the F₁ generation, and enhanced photic sensitivity of the circadian pacemaker mediating entrainment of oviposition rhythm of the strains from lower latitudes as compared to those from the higher latitudes.     

Preference for ethanol in feeding and oviposition in temperate and tropical populations of Drosophila melanogaster

The natural habitat of Drosophila melanogaster Meigen (Diptera: Drosophilidae) is fermenting fruits, which can be rich in ethanol. For unknown reasons, temperate populations of this cosmopolitan species have higher ethanol resistance than tropical populations. To determine whether this difference is accompanied by a parallel difference in preference for ethanol, we compared two European and two tropical African populations in feeding and oviposition preference for ethanol‐supplemented medium. Although females of all populations laid significantly more eggs on medium with ethanol than on control medium, preference of European females for ethanol increased as ethanol concentration increased from 2 to 6%, whereas that of African females decreased. In feeding tests, African females preferred control medium over medium with 4% ethanol, whereas European females showed no preference. Males of all populations strongly preferred control medium. The combination of preference for ethanol in oviposition, and avoidance or neutrality in feeding, gives evidence that adults choose breeding sites with ethanol for the benefit of larvae, rather than for their own benefit. The stronger oviposition preference for ethanol of temperate than tropical females suggests that this benefit may be more important in temperate populations. Two possible benefits of ethanol for which there is some experimental evidence are cryoprotection and protection against natural enemies.     

Persistence of orientation toward a temporarily invisible landmark in Drosophila melanogaster

In arena experiments with the walking fruit fly, we found a remarkable persistence of orientation toward a landmark that disappeared during the fly's approach. The directional stability achieved by `after-fixation' allows a fly to continue pursuit under natural conditions, where a selected target is frequently concealed by surrounding structures. The persistence of after-fixation was investigated in Buridan's paradigm, where a fly walks persistently back and forth between two inaccessible landmarks. Upon disappearance of a selected target, the flies maintained their intended course for more than 15 body lengths of approximately 2.5 mm in about 50% of the trials. About 13% even exceeded 75 body lengths. About 88% of the approaches clustered in equal portions around peaks at 2.4 s and 8.6 s. About 12% of the approaches persisted even longer. In contrast, a single peak at about 2.2 s is sufficient to describe the persistence of orientation in a random walk. The ability to pursue an invisible landmark is disturbed neither by a transient angular deviation from the course toward this landmark, when this target disappeared, nor by a distracting second landmark. Accordingly, after-fixation seems to require an internal representation of the direction toward the concealed target, and idiothetical course control to maintain this direction. Accepted: 19 September 1997     

Associations between female remating behavior, oogenesis and oviposition in Drosophila melanogaster and Drosophila pseudoobscura

An association between female remating behavior, oogenesis and oviposition was examined in Drosophila melanogaster and Drosophila pseudoobscura to investigate mechanisms that elicit remating. Females receptive to remating oviposited more eggs in both species; however, the species differed in the association between remating behavior and the number and distribution of oocyte stages. We found no differences in the number of either developing eggs of different stages or mature eggs between female D. pseudoobscura that were either receptive or nonreceptive to remating. In contrast, D. melanogaster females that are receptive to remating had significantly more mature eggs in the ovaries than nonreceptive females. Nonremating females had a significantly greater number of immature, vitellogenic oocytes. These results suggest that factors associated with oogenesis are related to female remating behavior in D. melanogaster but not in D. pseudoobscura. We discuss these results in conjunction with other evidence on the role male ejaculatory components play in mediating female remating behavior.     

Developmental plasticity of the locomotor activity rhythm of Drosophila melanogaster

We used four replicate outbred populations of Drosophila melanogaster to investigate whether the light regimes experienced during the pre-adult (larval and pupal) and early adult stages influence the free-running period (τ_DD) of the circadian locomotor activity rhythm of adult flies. In a series of two experiments four different populations of flies were raised from egg to eclosion in constant light (LL), in light/dark (LD) 12:12 h cycle, and in constant darkness (DD). In the first experiment the adult male and female flies were directly transferred into DD and their locomotor activity was monitored, while in the second experiment the locomotor activity of the emerging adult flies was first assayed in LD 12:12 h for 15 days and then in DD for another 15 days. The τ_DD of the locomotor activity rhythm of flies that were raised in all the three light regimes, LL, LD 12:12 h and in DD was significantly different from each other. The τ_DD of the locomotor activity rhythm of the flies, which were raised in DD during their pre-adult stages, was significantly shorter than that of flies that were raised as pre-adults in LL regime, which in turn was significantly shorter than that of flies raised in LD 12:12 h regime. This pattern was consistent across both the experiments. The results of our experiments serve to emphasise the fact that in order to draw meaningful inferences about circadian rhythm parameters in insects, adequate attention should be paid to control and specify the environment in which pre-adult rearing takes place. The pattern of pre-adult and early adult light regime effects that we see differs from that previously observed in studies of mutant strains of D. melanogaster, and therefore, also points to the potential importance of inter-strain differences in the response of circadian organisation to external influences.     

Temperature entrainment of the circadian cuticle deposition rhythm in Drosophila melanogaster

The cuticle deposition rhythm, which is observed in the apodeme of the furca in the thorax, is controlled by a peripheral circadian clock in the epidermal cells and entrained to light-dark (LD) cycles via CRYPTOCHROME (CRY) in Drosophila melanogaster. In the present study, we examined the effects of temperature (TC) cycles and the combination of LD and TC cycles on entrainment of the cuticle deposition rhythm. The rhythm was entrained to TC cycles, whose period was 28 h. In T = 21 and 24 h, the rhythm was entrained to TC cycles in some individuals. CRY is not necessary for temperature entrainment of the cuticle deposition rhythm because the rhythm in cry(b) (lacking functional CRY) was entrained to TC cycles. Temperature entrainment of the rhythm was achieved even when the thoraxes or furcae were cultured in vitro, suggesting that the mechanism for temperature entrainment is independent of the central clock in the brain and the site of the thermoreception resides in the epidermal cells. When LD and TC cycles with different periods were applied, the rhythm was entrained to LD cycles with a slight influence of TC cycles. Thus, the LD cycle is a stronger zeitgeber than the TC cycle. The variance of the number of the cuticle layers decreased in the flies kept under LD and TC cycles with the same period in which the thermophase coincided with the photophase. Therefore, we conclude that LD and TC cycles synergistically entrain the rhythm. Synergistic effects of LD and TC cycles on entrainment were also observed even when the thoraxes were cultured in vitro, suggesting that the light and temperature information is integrated within the peripheral circadian system.     

Setting the clock--by nature: circadian rhythm in the fruitfly Drosophila melanogaster

Nowadays humans mainly rely on external, unnatural clocks such as of cell phones and alarm clocks--driven by circuit boards and electricity. Nevertheless, our body is under the control of another timer firmly anchored in our genes. This evolutionary very old biological clock drives most of our physiology and behavior. The genes that control our internal clock are conserved among most living beings. One organism that shares this ancient clock mechanism with us humans is the fruitfly Drosophila melanogaster. Since it turned out that Drosophila is an excellent model, it is no surprise that its clock is very well and intensely investigated. In the following review we want to display an overview of the current understanding of Drosophila's circadian clock.     

Reassessment of the effect of biological rhythm mutations on learning in Drosophila melanogaster

A link between learning deficits and circadian period-lengthening mutations in Drosophila melanogaster previously has been reported. Mutant long-period males performed poorly in two learning assays involving experience-dependent courtship inhibition. In one, normal males that have courted fertilized females subsequently show courtship inhibition with virgin females. In the other, normal males that have courted sexually immature males subsequently fail to court other immature males. Those results have been reassessed in an extended study of genetic variants involving the period gene. 1. Long-period perL1 males demonstrated poor conditioned courtship inhibition when exposed to fertilized females; they showed normal courtship conditioning when exposed to immature males. This could be due to a perL1-associated olfactory deficit with fertilized females, since perL1 males were unable to discriminate behaviorally between fertilized and virgin females. 2. Other long-period males, including perL2 males and transgenic perL1 males bearing a truncated form of the per+ gene, were conditioned normally by fertilized females. Thus, the courtship inhibition defect is specific to the perL1 mutant strain. 3. perL1 (and other per mutant) flies showed normal acquisition and retention of a classically conditioned olfactory avoidance response. 4. Results from a new conditioned courtship inhibition experiment are presented; males exposed to fertilized females during training showed further courtship inhibition during subsequent exposure to fertilized females. From the perspective of learning theory, this can be viewed as a savings experiment.