Drosophila olfactory response rhythms require clock genes but not pigment dispersing factor or lateral neurons

Odors elicit a number of behavioral responses, including attraction and repulsion in Drosophila. In this study, the authors used a T-maze apparatus to show that wild-type Drosophila melanogaster exhibit a robust circadian rhythm in the olfactory attractive and repulsive responses. These responses were lower during the day and began to rise at early night, peaking at about the middle of the night and then declining thereafter. They were also independent of locomotor activity. The olfactory response rhythms were lost in period or timeless mutant flies (per0, tim0), indicating that clock genes control circadian rhythms of olfactory behavior. The rhythms in olfactory response persisted in the absence of the pigment-dispersing factor neuropeptide or the central pacemaker lateral neurons known to drive circadian patterns of locomotion and eclosion. These results indicate that the circadian rhythms in olfactory behavior in Drosophila are driven by pacemakers that do not control the rest-activity cycle and are likely in the antennae.     

Drosophila cryb mutation reveals two circadian clocks that drive locomotor rhythm and have different responsiveness to light

Cryptochrome (CRY) is a blue-light-absorbing protein involved in the photic entrainment of the circadian clock in Drosophila melanogaster. We have investigated the locomotor activity rhythms of flies carrying cryb mutant and revealed that they have two separate circadian oscillators with different responsiveness to light. When kept in constant light conditions, wild-type flies became arrhythmic, while cryb mutant flies exhibited free-running rhythms with two rhythmic components, one with a shorter and the other with a longer free-running period. The rhythm dissociation was dependent on the light intensities: the higher the light intensities, the greater the proportion of animals exhibiting the two oscillations. External photoreceptors including the compound eyes and the ocelli are the likely photoreceptors for the rhythm dissociation, since rhythm dissociation was prevented in so1;cryb and norpAP41;cryb double mutant flies. Immunohistochemical analysis demonstrated that the PERIOD expression rhythms in ventrally located lateral neurons (LNvs) occurred synchronously with the shorter period component, while those in the dorsally located per-expressing neurons showed PER expression most likely related to the longer period component, in addition to that synchronized to the LNvs. These results suggest that the Drosophila locomotor rhythms are driven by two separate per-dependent clocks, responding differentially to constant light.     

The period gene of Drosophila carries species-specific behavioral instructions.

We have analyzed and compared the circadian locomotor activity rhythms of Drosophila melanogaster and D.pseudoobscura. The rhythms of D.pseudoobscura are stronger and the periods shorter than those of D.melanogaster. We have also transformed D.melanogaster flies with a hybrid gene containing the coding region of the D.pseudoobscura period (per) gene. Behavioral assays of flies containing this hybrid gene show that the per protein encoded by the D.pseudoobscura per gene is able to rescue the rhythmic deficiencies of arrhythmic, pero1 D.melanogaster. More important, the rhythms of some of these strains are stronger and the periods shorter than those of D.melanogaster (and those of transformants which carry the equivalent D.melanogaster per gene construct) and hence resemble those of D.pseudoobscura. The results suggest that the primary amino acid sequence of the per gene encodes species-specific behavioral instructions that are detectable when only the per gene is transferred to a different species.     

Circadian consequence of socio-sexual interactions in fruit flies Drosophila melanogaster

In fruit flies Drosophila melanogaster, courtship is an elaborate ritual comprising chasing, dancing and singing by males to lure females for mating. Courtship interactions peak in the night and heterosexual couples display enhanced nighttime activity. What we do not know is if such socio-sexual interactions (SSI) leave long-lasting after-effects on circadian clock(s). Here we report the results of our study aimed at examining the after-effects of SSI (as a result of co-habitation of males and females in groups) between males and females on their circadian locomotor activity rhythm. Males undergo reduction in the evening activity peak and lengthening of circadian period, while females show a decrease in overall activity. Such after-effects, at least in males, require functional circadian clocks during SSI as loss-of-function clock mutants and wild type flies interacting under continuous light (LL), do not display them. Interestingly, males with electrically silenced Pigment Dispersing Factor (PDF)-positive ventral lateral (LNv) clock neurons continue to show SSI mediated reduction in evening activity peak, suggesting that the LNv clock neurons are dispensable for SSI mediated after-effects on locomotor activity rhythm. Such after-effects in females may not be clock-dependent because clock manipulated females with prior exposure to males show decrease in overall activity, more or less similar to rhythmic wild type females. The expression of SSI mediated after-effects requires a functional olfactory system in males because males with compromised olfactory ability do not display them. These results suggest that SSI causes male-specific, long-lasting changes in the circadian clocks of Drosophila, which requires the presence of functional clocks and intact olfactory ability in males.     

Non-ventral lateral neuron-based, non-PDF-mediated clocks control circadian egg-laying rhythm in Drosophila melanogaster

The authors report the results of their study aimed at investigating the consequence of targeted ablation of ventral lateral neurons (LN(v)s--neurons regulating eclosion and locomotor activity rhythms) and genetic disruption of pigment-dispersing factor (PDF--an important output of circadian clocks) on the egg-laying rhythm of Drosophila melanogaster. The results clearly suggest that genetic ablation of LN(v)s and loss of function mutation of PDF abolish eclosion and locomotor activity rhythms, whereas the egg-laying rhythm continues unabated. Furthermore, the results also demonstrate that the period of egg-laying rhythm remains unchanged under different ambient temperatures and nutrition levels, suggesting that the egg-laying rhythm of D. melanogaster is temperature and nutrition compensated. Based on these results, the authors conclude that the egg-laying rhythm in D. melanogaster is regulated by non-LN(v)-based, non-PDF-mediated circadian clocks.     

Temperature can entrain egg laying rhythm of Drosophila but may not be a stronger zeitgeber than light

In Drosophila multiple circadian oscillators and behavioral rhythms are known to exist, yet most previous studies that attempted to understand circadian entrainment have focused on the activity/rest rhythm and to some extent the adult emergence rhythm. Egg laying behavior of Drosophila females also follows circadian rhythmicity and has been seen to deviate substantially from the better characterized rhythms in a few aspects. Here we report the findings of our study aimed at evaluating how circadian egg laying rhythm in fruit flies Drosophila melanogaster entrains to time cues provided by light and temperature. Previous studies have shown that activity/rest rhythm of flies entrains readily to light/dark (LD) and temperature cycles (TC). Our present study revealed that egg laying rhythm of a greater percentage of females entrains to TC compared to LD cycles. Therefore, in the specific context of our study this result can be taken to suggest that egg laying clocks of D. melanogaster entrains to TC more readily than LD cycles. However, when TC were presented along with out-of-phase LD cycles, the rhythm displayed two peaks, one occurring close to lights-off and the other near the onset of low temperature phase, indicating that upon entrainment by TC, LD cycles may be able to exert a greater influence on the phase of the rhythm. These results suggest that temperature and light associatively entrain circadian egg laying clocks of Drosophila.     

Phase-shifting the fruit fly clock without cryptochrome

The blue light photopigment cryptochrome (CRY) is thought to be the main circadian photoreceptor of Drosophila melanogaster. Nevertheless, entrainment to light-dark cycles is possible without functional CRY. Here, we monitored phase response curves of cry(01) mutants and control flies to 1-hour 1000-lux light pulses. We found that cry(01) mutants phase-shift their activity rhythm in the subjective early morning and late evening, although with reduced magnitude. This phase-shifting capability is sufficient for the slowed entrainment of the mutants, indicating that the eyes contribute to the clock's light sensitivity around dawn and dusk. With longer light pulses (3 hours and 6 hours), wild-type flies show greatly enhanced magnitude of phase shift, but CRY-less flies seem impaired in the ability to integrate duration of the light pulse in a wild-type manner: Only 6-hour light pulses at circadian time 21 significantly increased the magnitude of phase advances in cry(01) mutants. At circadian time 15, the mutants exhibited phase advances instead of the expected delays. These complex results are discussed.     

Circadian regulation of egg-laying behavior in fruit flies Drosophila melanogaster

Significant progress has been made in our understanding of the neurogenetics of circadian clocks in fruit flies Drosophila melanogaster. Several pacemaker neurons and clock genes have now been identified and their roles in the cellular and molecular clockwork established. Some recent findings suggest that the basic architecture of the clock is multi-oscillatory; the clock mechanisms in the ventral lateral neurons (LN(v)s) of the fly brain govern locomotor activity and adult emergence rhythms, while the peripheral oscillators located in antennal cells regulate olfactory rhythm. Among circadian phenomena exhibited by Drosophila, the egg-laying rhythm is unique in many ways: (i) this rhythm persists under constant light (LL), while locomotor activity and adult emergence become arrhythmic, (ii) its circadian periodicity is much longer than 24h, and (iii) while egg-laying is rhythmic under constant darkness, the expression of two core clock genes period (per) and timeless (tim), is non-oscillatory in the ovaries. In this paper, we review our current knowledge of the circadian regulation of egg-laying behavior in Drosophila, and provide some possible explanations for its self-sustained nature. We conclude by discussing the existing limitations in our understanding of the regulatory mechanisms and propose few approaches to address them.     

Drosophila melanogaster deficient in protein kinase A manifests behavior-specific arrhythmia but normal clock function.

Drosophila melanogaster bearing mutations in the DCO gene, which encodes the major catalytic subunit of cAMP-dependent protein kinase (PKA), displays arrhythmic locomotor activity strongly suggesting a role for PKA in the circadian timing system. This arrhythmicity might result from a requirement for PKA activity in photic resetting pathways, the timekeeping mechanism itself, or downstream effector pathways controlling overt behavioral rhythms. To address these possibilities, we examined the protein and mRNA products from the clock gene period (per) in PKA-deficient flies. The per protein (PER) and mRNA products undergo daily cycles in the heads and bodies of DCO mutants that are indistinguishable from those observed in control wild-type flies. These results indicate that PKA deficiencies affect the proper functioning of elements downstream of the Drosophila timekeeping mechanism. The requirement for PKA in the manifestation of rhythmic activity was preferentially greater in the absence of environmental cycles. However, PKA does not appear to play a universal role in output functions because the clock-controlled eclosion rhythm is normal in DCO mutants. Our results suggest that PKA plays a critical role in the flow of temporal information from circadian pacemaker cells to selective behaviors.     

Separate sets of cerebral clock neurons are responsible for light and temperature entrainment of Drosophila circadian locomotor rhythms

The fruit fly Drosophila melanogaster shows a bimodal circadian locomotor rhythm with peaks at lights-on and before lights-off, which are regulated by multiple clocks in the brain. Even under light-dark cycles, the timing of the evening peak is highly dependent on temperature, starting earlier under lower ambient temperature but terminating almost at the same time. In the present study, using behavioral and immunohistochemical assays, the authors show that separate groups of clock neurons, either light-entrainable or temperature-entrainable, form a functional system driving the locomotor rhythm. When subjected to a light cycle combined with a temperature cycle advanced by 6 h relative to the light cycle, the dorsally located neurons (DNs) and lateral posterior neurons (LPNs) shifted their phase of TIMELESS expression, but the laterally located protocerebral neurons (LNs) basically maintained their original phase. Thus, the LNs seem to be preferentially light-entrainable and the DNs and LPNs to be primarily temperature-entrainable. In pdf(01) mutant flies that lack the neuropeptide PDF in the ventral groups of LNs, the onset of the evening peak was greatly advanced even under synchronized light and temperature cycles and was shifted even more than in wild-type flies in response to a 6-h phase shift of the temperature cycle, suggesting that ventral LNs have a strong impact on the phase of the other cells. It seems likely that the 2 sets of clock cells with different entrainability to light and temperature, and the coupling between them, enable Drosophila to keep a proper phase relationship of circadian activity with respect to the daily light and temperature cycles.     

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.     

Circadian phenotypes of Drosophila fragile x mutants in alternative genetic backgrounds

Drosophila FMR1 mutants are models of human fragile X syndrome. They show a loss of locomotor activity rhythm and severe degradation of eclosion timing. We analyzed the circadian behavior of FMR1 mutants (dfmr1(B55)) in two genetic backgrounds, yellow white (yw) and Canton S (CS). The arrhythmic phenotype of circadian locomotor activity in constant darkness (DD) did not significantly change in either genetic background. Surprisingly, eclosion timing was completely restored by backcrossing dfmr1(B55) with yw or CS flies. Morphological analysis of the small ventrally located lateral neurons of FMR1 mutants revealed that the dorsal-projection area was significantly larger in arrhythmic than rhythmic flies. In addition, dfmr1(B55) mutants in both genetic backgrounds had a significantly lower evening peak in the light-dark (LD) cycle. These results indicate that lack of FMR1 does not affect eclosion timing, but alters locomotor activity patterns in both LD and DD conditions by affecting the arborization of small ventrally located lateral neurons. Thus, the FMR1 gene may regulate the circadian-related locomotor activity of Drosophila.     

Casein kinase I epsilon does not rescue double-time function in Drosophila despite evolutionarily conserved roles in the circadian clock

Double-time (dbt) is a casein kinase gene involved in cell survival, proliferation, and circadian rhythms in the fruit fly, Drosophila melanogaster. Genetic and biochemical studies have shown that dbt and its mammalian ortholog casein kinase I epsilon (hckI epsilon) regulate the circadian phosphorylation of period (per), thus controlling per subcellular localization and stability. Mutations in these kinases can shorten the circadian period in both mammals and Drosophila. Since similar activities in circadian clock have been described for these kinases, we investigated whether the expression of mammalian casein kinase I can replace the activity of dbt in flies. Global expression of the full-length dbt rescued lethality of the null mutant dbt revVIII and rescued flies showed normal locomotor activity rhythms. Global expression of dbt also restored the locomotor activity rhythm of the arrhythmic genotype, dbt ar/dbt revVIII. In contrast, global expression of hckI epsilon or hckI alpha did not rescue lethality or locomotor activity of dbt mutants. Furthermore dbt overexpression in wild-type clock cells had only a small effect on period length, whereas hckI epsilon expression in clock cells greatly lengthened period to ~30.5 hours and increased the number of arrhythmic flies. These results indicate that hckI epsilon cannot replace the activity of dbt in flies despite the high degree of similarity in primary sequence and kinase function. Moreover, expression of hck Iepsilon in flies appears to interfere with dbt activity. Thus, caution should be used in interpreting assays that measure activity of mammalian casein kinase mutants in Drosophila, or that employ vertebrate CKI in studies of dPER phosphorylations.     

Insect circadian rhythms and photoperiodism

Two clock-controlled processes, overt circadian rhythmicity and the photoperiodic induction of diapause, are described in the blow fly,Calliphora vicina and the fruit fly,Drosophila melanogaster. Circadian locomotor rhythms of the adult flies reflect endogenous, self-sustained oscillations with a temperature compensated period. The free-running rhythms become synchronised (entrained) to daily light:dark cycles, but become arrhythmic in constant light above a certain intensity. Some flies show fragmented rhythms (internal desynchronisation) suggesting that overt rhythmicity is the product of a multioscillator (multicellular) system. Photoperiodic induction of larval diapause inC. vicina and of ovarian diapause inD. melanogaster is also based on the circadian system but seems, to involve a separate mechanism at both the molecular and neuronal levels. For both processes in both species, the compound eyes and ocelli are neither essential nor necessary for photic entrainment, and the circadian clock mechanism is not within the optic lobes. The central brain is the most likely site for both rhythm generation and extra-optic photoreception. InD. melanogaster, a group of lateral brain neurons has been identified as important circadian pacemaker cells, which are possibly also photo-sensitive. Similar lateral brain neurons, staining for arrestin, a protein in the phototransduction ‘cascade’ and a selective marker for photoreceptors in both vertebrates and invertebrates, have been identified inC. vicina. Much less is known about the cellular substrate of the photoperiodic mechanism, but this may involve thepars intercerebralis region of the mid-brain.     

Temporal mating isolation driven by a behavioral gene in Drosophila

Speciation is the evolutionary process in which new barriers to gene exchange are created. These barriers may be physical, leading to spatial separation of subpopulations and resulting in allopatric speciation, or they may be temporal, giving rise to allochronic speciation, and may include the time of day or the time of year when mating takes place. Drosophila melanogaster and D. pseudoobscura show different temporal patterns of circadian locomotor activity that are determined by the circadian clock gene period (per). Genes that control aspects of behavior that might be relevant to courtship and mating, such as locomotor patterns, become obvious candidates for involvement in the speciation process. However, evidence for the role of individual genes in the mechanism of mate choice has proved elusive. We have used transgenic flies carrying the natural per genes from these two Drosophila species to reveal that per has the potential to provide the permissive conditions for speciation, by affecting mate choice through a mechanism involving the species-specific timing of mating behavior.     

Drosophila ebony activity is required in glia for the circadian regulation of locomotor activity

Previous studies suggest that glia may be required for normal circadian behavior, but glial factors required for rhythmicity have not been identified in any system. We show here that a circadian rhythm in Drosophila Ebony (N-beta-alanyl-biogenic amine synthetase) abundance can be visualized in adult glia and that glial expression of Ebony rescues the altered circadian behavior of ebony mutants. We demonstrate that molecular oscillator function and clock neuron output are normal in ebony mutants, verifying a role for Ebony downstream of the clock. Surprisingly, the ebony oscillation persists in flies lacking PDF neuropeptide, indicating it is regulated by an autonomous glial oscillator or another neuronal factor. The proximity of Ebony-containing glia to aminergic neurons and genetic interaction results suggest a function in dopaminergic signaling. We thus suggest a model for ebony function wherein Ebony glia participate in the clock control of dopaminergic function and the orchestration of circadian activity rhythms.     

Conserved regions of the timeless (tim) clock gene in Drosophila analyzed through phylogenetic and functional studies.

Circadian (approximately 24-hr) rhythms in Drosophila melanogaster depend upon cyclic expression of the period (per) and timeless (tim) genes, which encode interacting components of the endogenous clock. The per gene has been isolated from other insects and, more recently, a per ortholog was found in mammals where its expression oscillates in a circadian fashion. We report here the complete sequence of a tim gene from another species, Drosophila virilis. TIM is better conserved than the PER protein is between these two species (76 vs. 54% overall amino acid identity), and putative functional domains, such as the PER interaction domains and the nuclear localization signal, are highly conserved. The acidic domain and the cytoplasmic localization domain, however, are within the least conserved regions. In addition, the initiating methionine in the D. virilis gene lies downstream of the proposed translation start for the original D. melanogaster tim cDNA and corresponds to the one used by D. simulans and D. yakuba. Among the most conserved parts of TIM is a region of unknown function near the N terminus. We show here that deletion of a 32 amino acid segment within this region affects rescue of rhythms in arrhythmic tim01 flies. Flies carrying a full-length tim transgene displayed rhythms with approximately 24-hr periods, indicating that a fully functional clock can be restored in tim01 flies through expression of a tim transgene. Deletion of the segment mentioned above resulted in very long activity rhythms with periods ranging from 30.5 to 48 hr.     

Circadian changes in Drosophila motor terminals

In Drosophila melanogaster, as in most other higher organisms, a circadian clock controls the rhythmic distribution of rest/sleep and locomotor activity. Here we report that the morphology of Drosophila flight neuromuscular terminals changes between day and night, with a rhythm in synaptic bouton size that continues in constant darkness, but is abolished during aging. Furthermore, arrhythmic mutations in the clock genes timeless and period also disrupt this circadian rhythm. Finally, these clock mutants also have an opposing effect on the nonrhythmic phenotype of neuronal branching, with tim mutants showing a dramatic hyperbranching morphology and per mutants having fewer branches than wild-type flies. These unexpected results reveal further circadian as well as nonclock related pleiotropic effects for these classic behavioral mutants.     

Circadian clock of Drosophila montana is adapted to high variation in summer day lengths and temperatures prevailing at high latitudes

Photoperiodic regulation of the circadian rhythms in insect locomotor activity has been studied in several species, but seasonal entrainment of these rhythms is still poorly understood. We have traced the entrainment of activity rhythm of northern Drosophila montana flies in a climate chamber mimicking the photoperiods and day and night temperatures that the flies encounter in northern Finland during the summer. The experiment was started by transferring freshly emerged females into the chamber in early and late summer conditions to obtain both non-diapausing and diapausing females for the studies. The locomotor activity of the females and daily changes in the expression levels of two core circadian clock genes, timeless and period, in their heads were measured at different times of summer. The study revealed several features in fly rhythmicity that are likely to help the flies to cope with high variation in the day length and temperature typical to northern summers. First, both the non-diapausing and the diapausing females showed evening activity, which decreased towards the short day length as observed in the autumn in nature. Second, timeless and period genes showed concordant daily oscillations and seasonal shifts in their expression level in both types of females. Contrary to Drosophila melanogaster, oscillation profiles of these genes were similar to each other in all conditions, including the extremely long days in early summer and the cool temperatures in late summer, and their peak expression levels were not locked to lights-off transition in any photoperiod. Third, the diapausing females were less active than the non-diapausing ones, in spite of their younger age. Overall, the study showed that D. montana clock functions well under long day conditions, and that both the photoperiod and the daily temperature cycles are important zeitgebers for seasonal changes in the circadian rhythm of this species.