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.     

Cyclic presence and absence of conspecifics alters circadian clock phase but does not entrain the locomotor activity rhythm of the fruit fly Drosophila melanogaster

Circadian clocks use a wide range of environmental cues, including cycles of light, temperature, food, and social interactions, to fine-tune rhythms in behavior and physiology. Although social cues have been shown to influence circadian clocks of a variety of organisms including the fruit fly Drosophila melanogaster, their mechanism of action is still unclear. Here, the authors report the results of their study aimed at investigating if daily cycles of presence and absence (PA) of conspecific male visitors are able to entrain the circadian locomotor activity rhythm of male hosts living under constant darkness (DD). The results suggest that PA cycles may not be able to entrain circadian locomotor activity rhythms of Drosophila. The outcome does not change when male hosts are presented with female visitors, suggesting that PA cycles of either sex may not be effective in bringing about stable entrainment of circadian clocks in D. melanogaster. However, in hosts whose clock phase has already been set by light/dark (LD) cycles, daily PA cycles of visitors can cause measurable change in the phase of subsequent free-running rhythms, provided that their circadian clocks are labile. Thus, the findings of this study suggest that D. melanogaster males may not be using cyclic social cues as their primary zeitgeber (time cue) for entrainment of circadian clocks, although social cues are capable of altering the phase of their circadian rhythms.     

鱼藤酮对果蝇运动行为及果蝇头部多巴胺合成相关酶基因表达的影响

为了探讨鱼藤酮对黑腹果蝇Drosophila melanogaster运动行为的影响与其头部多巴胺水平之间的关系,我们测定了鱼藤酮对黒腹果蝇成虫运动行为、头部多巴胺水平及酪氨酸羟化酶和多巴脱羧酶基因表达的影响。结果表明：与取食未加入药剂饲料的果蝇相比雌成虫用0.2～0.8 mmol/L、雄成虫用0.1～0.8 mmol/L浓度药液配制的饲料连续饲养6 d后运动能力显著下降,在0.8 mmol/L浓度下雌、雄成虫的运动能力分别仅为对照的55.6%和49.1%。取食用0.8 mmol/L浓度药液配制饲料6,12和21 d的果蝇雌、雄成虫头部多巴胺水平均显著下降,雌成虫头部多巴胺水平分别为对照雌成虫的83.2%,72.3%和59.8%;雄成虫头部多巴胺水平分别为对照雄成虫的79.3%,66.8%和53.2%。用0.8 mmol/L浓度鱼藤酮处理6,12和21d,雌成虫头部酪氨酸羟化酶基因（pale）的表达水平分别为对照的76.3%,51.4%和37.3%,多巴脱羧酶基因（Ddc）的表达水平分别为对照的87.1%,78.2%和63.5%, 均显著下降。结果提示,鱼藤酮可干扰果蝇成虫头部酪氨酸羟化酶和多巴脱羧酶基因的表达,导致果蝇头部多巴胺水平下降,进而影响了果蝇的运动行为。     

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.     

Larval ethanol exposure alters adult circadian free-running locomotor activity rhythm in Drosophila melanogaster

Alcohol consumption causes disruptions in a variety of daily rhythms, including the sleep-wake cycle. Few studies have explored the effect of alcohol exposure only during developmental stages preceding maturation of the adult circadian clock, and none have examined the effects of alcohol on clock function in Drosophila. This study investigates developmental and behavioral correlates between larval ethanol exposure and the adult circadian clock in Drosophila melanogaster, a well-established model for studying circadian rhythms and effects of ethanol exposure. We reared Drosophila larvae on 0%, 10%, or 20% ethanol-supplemented food and assessed effects upon eclosion and the free-running period of the circadian rhythm of locomotor activity. We observed a dose-dependent effect of ethanol on period, with higher doses resulting in shorter periods. We also identified the third larval instar stage as a critical time for the developmental effects of 10% ethanol on circadian period. These results demonstrate that developmental ethanol exposure causes sustainable shortening of the adult free-running period in Drosophila melanogaster, even after adult exposure to ethanol is terminated, and suggests that the third instar is a sensitive time for this effect.     

Haltere-mediated equilibrium reflexes of the fruit fly, Drosophila melanogaster.

Flies display a sophisticated suite of aerial behaviours that require rapid sensory-motor processing. Like all insects, flight control in flies is mediated in part by motion-sensitive visual interneurons that project to steering motor circuitry within the thorax. Flies, however, possess a unique flight control equilibrium sense that is encoded by mechanoreceptors at the base of the halteres, small dumb-bell-shaped organs derived through evolutionary transformation of the hind wings. To study the input of the haltere system onto the flight control system, I constructed a mechanically oscillating flight arena consisting of a cylindrical array of light-emitting diodes that generated the moving image of a 30 degrees vertical stripe. The arena provided closed-loop visual feedback to elicit fixation behaviour, an orientation response in which flies maintain the position of the stripe in the front portion of their visual field by actively adjusting their wing kinematics. While flies orientate towards the stripe, the entire arena was swung back and forth while an optoelectronic device recorded the compensatory changes in wing stroke amplitude and frequency. In order to reduce the background changes in stroke kinematics resulting from the animal's closed-loop visual fixation behaviour, the responses to eight identical mechanical rotations were averaged in each trial. The results indicate that flies possess a robust equilibrium reflex in which angular rotations of the body elicit compensatory changes in both the amplitude and stroke frequency of the wings. The results of uni- and bilateral ablation experiments demonstrate that the halteres are required for these stability reflexes. The results also confirm that halteres encode angular velocity of the body by detecting the Coriolis forces that result from the linear motion of the haltere within the rotating frame of reference of the fly's thorax. By rotating the flight arena at different orientations, it was possible to construct a complete directional tuning map of the haltere-mediated reflexes. The directional tuning of the reflex is quite linear such that the kinematic responses vary as simple trigonometric functions of stimulus orientation. The reflexes function primarily to stabilize pitch and yaw within the horizontal plane.     

Cation-distribution in developing follicles of the fruit fly Drosophila melanogaster

Abstract. Cations were precipitated with potassium antimonate in ovarian follicles of Drosophila and the distribution of the formed precipitates was studied. The precipitates were analyzed with a laser microprobe mass analyzer (LAMMA) and found to contain a high concentration of calcium; potassium and sodium were also detected. On counting the antimon precipitates in stage 10B follicles with the electron microscope, few precipitates per unit area were found in anterior nurse cells, but more in posterior nurse cells; the highest precipitate density occurred consistently in the oocyte. When follicles of different stages were compared, the precipitate density was found to increase in the ooplasm and in the posterior nurse cells during vitellogenesis, whereas it remained nearly constant in the anterior nurse cells. Thus, the ratio of precipitates between the posterior and anterior end of the follicle increases during vitellogenesis. It begins to decrease at the time when the nurse cells collapse. These results suggest that the electrical polarity observed in polytrophic ovarioles may be based on differences in the cation distribution along the antero-posterior axis of the follicle.     

Temporal pattern of locomotor activity in Drosophila melanogaster

The temporal pattern of locomotor activity of single Drosophila melanogaster flies freely walking in small tubes is described. Locomotor activity monitored by a light gate has a characteristic time-course that depends upon age and the environmental conditions. Several methods are applied to assess the complexity of the temporal pattern. The pattern varies according to sex, genotype, age and environmental conditions (food; light). Activity occurs clustered in bouts. The intrinsic bout structure is quantified by four parameters: number of light gate passages (counts) per bout, duration of a bout, pause between two successive bouts and mean bout period. In addition, the distribution of the periods between light-gate crossings (inter-count intervals) as function of inter-count interval duration reveals a power law, suggesting that the overall distribution of episodes of activity and inactivity has a fractal structure. In the dark without food, the fractal dimension which represents a measure of the complexity of the pattern is sex, genotype and age specific. Fractality is abolished by additional sensory stimulation (food; light). We propose that time-course, bout structure and fractal dimension of the temporal pattern of locomotor activity describe different aspects of the fly's central pattern generator for locomotion and its motivational control. Accepted: 10 October 1998     

Limitation of size by hypoxia in the fruit fly Drosophila melanogaster

The size of an organism is of fundamental importance in all biological processes. It dictates many of the critical interactions and physical factors that delimit the envelope within which an organism can grow. We investigated the effects of reduced oxygen on size and development in the fruit fly Drosophila melanogaster, and showed that limiting the oxygen in the environment limits both whole animal and cell size. When oxygen levels were reduced from 20% in nitrogen to 15%, 10% and 7.5%, there was a linear decrease in both male and female mass. Both cell size and cell number decreased in low oxygen, but changes in cell size accounted for a larger proportion of the overall change in fly size. Cell numbers decreased by a maximum of 11% between flies reared in 20% oxygen and those reared in 7.5% oxygen, whereas cell surface area decreased by 17%. Low oxygen levels increased development time and mortality, but reduced fecundity. Reducing the level of oxygen available significantly slowed development times, with flies reared in 10% oxygen emerging on average 1.5 days later than those in 20% oxygen. The effect of oxygen on size is reversible during embryonic and larval development up to the pupal stage, when final size is set.     

Circadian rhythm of the locomotor activity in Drosophila melanogaster and its mutants 'sine oculis' and 'small optic lobes'

ABSTRACT. The locomotor activity patterns of wildtype Drosophila melanogaster and the mutants so (sine oculis) and sol (small optic lobes) were investigated. In all strains the proportions showing circadian rhythmicity, arrhythmicity and more complex patterns which could not be thus classified were similar. The occurrence of abnormal activity patterns is thus not a property of the mutation as previously claimed (Mack & Engelmann, 1981). In flies with a distinct circadian rhythmicity, the period lengths (τ) varied between strains, τ for wildtype Italy and the mutant so was longer than for wildtype Berlin and the mutant sol. As different τ's have been reported by others, τ does not seem to be closely determined for Drosophila melanogaster. Many flies exhibited two rhythms simultaneously, one with τ shorter and one with τ longer than 24 h, apparently implying two-oscillator control of the locomotor activity. The eyeless so mutants were entrained by LD cycles, so the compound eyes are not necessary, and so must possess the relevant photoreceptor(s) elsewhere. This may therefore also be true for the wildtype. Histology of the so adults revealed no correlation between the degree of reduction in the medulla and the occurrence of abnormal activity patterns. Since the only structures common to the medulla of so and sol are known to be large tangential cells, it is concluded that either they are of importance for the rhythmic system, or the oscillator(s) controlling locomotor activity is (are) not located in the medulla.     

Interaction between sexes and between different circadian phenotypes affects lifespan in the fruit fly Drosophila melanogaster

Circadian clocks regulate the daily temporal structure of physiological and behavioural functions. In the fruit fly Drosophila melanogaster Meigen, disruption of daily rhythms is suggested to reduce the fly's lifespan. In the present study, because pairs of mixed‐sex flies are known to show an activity pattern different from that of individual flies, this hypothesis is tested by measuring the lifespan of flies housed same‐sexually or mixed‐sexually under an LD 12 : 12 h photocycle at a constant temperature of 25 °C. The effect of housing wild‐type (Canton‐S) flies with period (per) circadian clock mutant flies is also examined because the mutant flies have different daily activity patterns. When males and females of wild‐type flies are housed together, their lifespan is substantially lengthened (males) or shortened (females) compared with same‐sex housed flies. The shortening of the lifespan in females is significantly enhanced when mated with per mutant males. The shortening effects are significantly reduced when the mixed‐sex interaction is limited for the first 5 days after emergence. A slight elongation in lifespan, rather than a reduction, occurs when wild‐type females are housed same‐sexually with per⁰ or per^L mutant flies. In male flies, the elongation of lifespan occurs not only when wild‐type males are housed with wild‐type, per⁰ or per^L females, but also when housed with per⁰ or per^S mutant males. Mixed‐sex couples always show altered daily locomotor rhythms with an enhanced night‐time activity, whereas same‐sex couples show daily behavioural profiles slightly altered but essentially similar to a sum of the respective two flies. No significant correlation is found between the lifespan and reproductive capacity. These results suggest that the alteration of daily activity rhythms and sexual interaction may have significant impact on the fly's lifespan.     

UDP-galactose 4'-epimerase activities toward UDP-Gal and UDP-GalNAc play different roles in the development of Drosophila melanogaster

In both humans and Drosophila melanogaster, UDP-galactose 4'-epimerase (GALE) catalyzes two distinct reactions, interconverting UDP-galactose (UDP-gal) and UDP-glucose (UDP-glc) in the final step of the Leloir pathway of galactose metabolism, and also interconverting UDP-N-acetylgalactosamine (UDP-galNAc) and UDP-N-acetylglucosamine (UDP-glcNAc). All four of these UDP-sugars serve as vital substrates for glycosylation in metazoans. Partial loss of GALE in humans results in the spectrum disorder epimerase deficiency galactosemia; partial loss of GALE in Drosophila melanogaster also results in galactose-sensitivity, and complete loss in Drosophila is embryonic lethal. However, whether these outcomes in both humans and flies result from loss of one GALE activity, the other, or both has remained unknown. To address this question, we uncoupled the two activities in a Drosophila model, effectively replacing the endogenous dGALE with prokaryotic transgenes, one of which (Escherichia coli GALE) efficiently interconverts only UDP-gal/UDP-glc, and the other of which (Plesiomonas shigelloides wbgU) efficiently interconverts only UDP-galNAc/UDP-glcNAc. Our results demonstrate that both UDP-gal and UDP-galNAc activities of dGALE are required for Drosophila survival, although distinct roles for each activity can be seen in specific windows of developmental time or in response to a galactose challenge. By extension, these data also suggest that both activities might play distinct and essential roles in humans.     

Influence of the corticosterone rhythm on photic entrainment of locomotor activity in rats

The question of involvement of glucocorticoid hormones as temporal signals for the synchronization of the timekeeping system was addressed in rats with different corticosterone status. The authors showed that adrenalectomy had no effects on the synchronization of wheel-running activity rhythms to a steady-state LD 12:12 cycle, regardless of whether it was compensated for by a corticosterone replacement therapy that either reinstated constant plasma concentrations of the hormone or mimicked its natural rhythm. However, after a 12-h phase shift (daylight reversal), the lack of circulating corticosterone induced a significant shortening of the resynchronization rate (less than 3 days vs. 7 days). Normalization required restoration of a rhythmic corticosterone secretion that was synchronized to the new photoperiod. Under constant darkness, the corticosterone rhythm did not show any synchronizing effect, providing evidence that it participates in entrainment of the locomotor activity rhythm through modulation of light effects. It is proposed that, under stable lighting conditions, circulating glucocorticoids contribute to stabilizing activity rhythms by reinforcing resistance of the circadian timing system to variations of the photoperiod. Experimental evidence that serotonergic neurons are involved in relaying their modulatory effects to the clock is also presented.     

A temperature-dependent timing mechanism is involved in the circadian system that drives locomotor rhythms in the fruit fly Drosophila melanogaster

The circadian clock of Drosophila melanogaster is thought to include rhythmic expression of period gene. Recent studies suggested, however, that a per-less oscillation is also involved in the regulation of circadian locomotor rhythms. In the present study, we examined the existence and the property of the possible per-less oscillation using arrhythmic clock mutant flies carrying per (01), tim(01), dClk(Jrk) or cyc(01), which lack rhythmic per expression. When temperature cycles consisting of 25 degrees C and 30 degrees C with various periods (T=8-32 hr) were given, wild-type (Canton-S) flies showed locomotor rhythms entrained to temperature cycles over a wide range of period (T=8-32 hr) in constant light (LL) while only to T=24 hr in constant darkness (DD). The mutant flies showed rhythms synchronizing with the given cycle both under LL and DD. In per(01) and tim(01) flies, the phase of a major peak slightly changed dependent on Ts in DD, while it did not in dClk(Jrk) and cyc(01) flies. When they were transferred from a constant temperature to a temperature cycle under DD, several cycles were necessary to establish a clear temperature entrainment in per(01) and tim (01) flies. These results suggest that per(01) and tim(01) flies have a temperature-entrainable weak oscillatory mechanism and that the per-less oscillatory mechanism may require dClk and cyc. In addition, per (01) and tim(01) flies changed from thermoactive in DD to cryoactive in LL, while dClk(Jrk) and cyc(01) flies did not. It is thus suggested that dClk and cyc are also involved in determining the light-associated temperature preference in per(01) and tim(01) flies.     

Dissociation of photoreceptors from whole heads of the fruit fly,Drosophila melanogaster

Photoreceptor cells that were mostly free of extracellular material and suitable for most electrophysiological study procedures were dissociated from whole heads of the fruit fly, Drosophila melanogaster, by a simple smash technique employing gentle chopping by a razor blade through Parafilm sheets. A variety of commonly available proteolytic and glycolytic digestion enzymes were tested as additions to the basic dissociation procedure described. With the aid of Nomarski interference contrast optics, periodic acid-Schiff staining, and fluorescent labeling and microscopy methods, it was determined that proteolytic enzymatic digestion does little to enhance the dissociation procedure, and instead, often damages the cells that one is attempting to recover. Unexpectedly, certain glycolytic enzymes, when added to the basic procedure, appear to enhance the recovery of intact viable Drosophila photoreceptors that are stripped of most extracellular material. Based on these results, a hypothesis concerning the biochemical nature of the extracellular matrix of the Drosophila retina is proposed. Drosophila photoreceptors are an interesting model system for the study of invertebrate phototransduction and photoreceptor cell biology because of their many well-characterized mutant strains. The technique described here should produce clean viable photoreceptors or ommatidia that respond to light, and that are suitable for patch clamping or cell culture.     

The morphology, physiology, and neural projections of supernumerary compound eyes in Drosophila melanogaster

Supernumerary compound eyes in Drosophila melanogaster produced by the extra eye (ee) mutation were analyzed with regard to their morphology, physiology, and neural projections. Electron and light microscopy revealed that large extra eyes often possess the normal complement of compound-eye cell types and that these cells usually have standard fine structure. In addition, the array of photoreceptor cell rhabdomeres within individual supernumerary ommatidia is standardly trapezoidal, and ommatidial subpopulations having mirror-image configurations of their rhabdomeric trapezoids are separated by an equator in extra eyes. Light stimulation of supernumerary eyes can elicit photoreceptor depolarization potentials as evidenced by electroretinographic recordings from them. In addition, extra-eye photoreceptor cells have a functional pupillary response to light stimulation. Although the supernumerary eyes can be functionally and anatomically standard, examination of serial, silver-stained sections of extra-eye heads has shown that their photoreceptor axons seldom innervate the brain. This situation obtains even in a case in which the normal, ipsilateral compound eye was removed by the eyeless mutation. In contrast, rare supernumerary antennae occasionally found in ee stocks have receptor cells whose axons innervate ventral brain. In addition to duplications of cuticular epithelia, extra glial cells, muscle fibers, and ocellar interneurons are sometimes found in extra-eye bearing flies. Discussion of these results focuses on a polarity guidance hypothesis which models the growth of adult photoreceptor axons into the brain during normal development.     

Evidence for adaptive male mate choice in the fruit fly Drosophila melanogaster

Theory predicts that males will benefit when they bias their mating effort towards females of higher reproductive potential, and that this discrimination will increase as males become more resource limited. We conducted a series of experiments to test these predictions in a laboratory population of the fruitfly, Drosophila melanogaster. In this species, courtship and copulation have significant costs to males, and females vary greatly in fecundity, which is positively associated with body size. When given a simultaneous choice between small and large virgin females, males preferentially mated with larger, more fecund, females. Moreover, after males had recently mated they showed a stronger preference for larger females. These results suggest that male D. melanogaster adaptively allocate their mating effort in response to variation in female quality and provide some of the first support for the theoretical prediction that male stringency in mate choice increases as resources become more limiting.     

Resource limitation and responses to rivals in males of the fruit fly Drosophila melanogaster

Diet has a profound direct and indirect effect on reproductive success in both sexes. Variation in diet quality and quantity can significantly alter the capacity of females to lay eggs and of males to deliver courtship. Here, we tested the effect of dietary resource limitation on the ability of male Drosophila melanogaster to respond adaptively to rivals by extending their mating duration. Previous work carried out under ad libitum diet conditions showed that males exposed to rivals prior to mating significantly extend mating duration, transfer more ejaculate proteins and achieve higher reproductive success. Such adaptive responses are predicted to occur because male ejaculate production may be limited. Hence, ejaculate resources require allocation across different reproductive bouts, to balance current vs. future reproductive success. However, when males suffer dietary limitation, and potentially have fewer reproductive resources to apportion, we expect adaptive allocation of responses to rivals to be minimized. We tested this prediction and found that males held on agar‐only diets for 5–7 days lost the ability to extend mating following exposure to rivals. Interestingly, extended mating was retained in males held on low yeast/sugar: no sugar/yeast diet treatments, but was mostly lost when males were maintained on ‘imbalanced’ diets in which there was high yeast: no sugar and vice versa. Overall, the results show that males exhibit adaptive responses to rivals according to the degree of dietary resource limitation and to the ratio of individual diet components.