Effects of Mutation or Deletion

per mutants are characterized by aberrant rhythms involving eclosion (hatching from the pupal case) and locomotor activity (Knopka, 1991). Mutation also affects:

  • 1) the rhythm of the larval heartbeat
  • 2) octopamine (a neurotransmitter) synthesis the brain with a corresponding decrease in the level of tyrosine decarboxylase
  • 3) fluctuation in membrane potentials in larval salivary glands
  • 4) the location of neural secretory cells in the brain
  • 5) the interval between pulses of wing vibration
  • 6) courtship song rhythms.

    Mosaic analysis of per mutations indicates that the circadian clock is located in the fly's head, whereas the courtship song resides in the fly's body, probably in the thoracic ganglion. Perhaps different per transcription of splice variants are responsible for these two functions (Citri, 1987).

    To identify new components of the Drosophila circadian clock, chemically mutagenized flies were screened for suppressors or enhancers of the long periods characteristic of the period mutant allele per. A mutant maps at the timeless gene. This novel allele, timSL, alters the temporal pattern of per protein nuclear localization and restores temperature compensation to per mutant flies. perL mutants exhibit a lengthening period of locomoter activity from 24 to 29 hours, and lack the ability to compensate photoperiod response to substantial changes in temperature. timSL (tim suppressor of perL mutation) more generally manifests specific interactions with different per alleles. The identification of this first period-altering timSL allele (previous tim mutants have been null) provides further evidence that TIM is a major component of the clock, and the allele-specific interactions with PER provide evidence that the PER/TIM heterodimer is a unit of circadian function. timSL alters the TIM phosphorylation pattern during the late night. The effects of timSL on TIM phosphorylation (TIM-SL is phosphorylated to a greater extent than wild-type TIM) suggest that timSL functions as a partial bypass suppressor of per and provide evidence that the TIM phosphorylation program contributes to the circadian timekeeping mechanism (Rutila, 1996).

    Light affects three features of the Period-Timeless program: PER and TIM phosphorylation, PER and TIM protein accumulation, and PER and TIM messenger RNA cycling. A post-transcriptional effect on the PER-TIM complex is the likely primary clock target, which then delays the subsequent decrease in PER and TIM mRNA levels. This is consistent with a negative feedback loop, in which the PER-TIM complex contributes to the decrease in PER and TIM mRNA levels, presumably at the transcriptional level. There are enhanced constant light effects on the perS mutant, which further support negative feedback as well as support its importance to entrainment of the flies to a 24 hour cycle, far from their intrinsic period of 19 hours. The perS mutant leads to a truncated protein accumulation phase and a subsequent premature perS mRNA increase. A standard 24 hour light-dark cycle delays the negative feedback circuit and extends the RNA and protein profiles, compensating for the accelerated mRNA increase and restoring the rhythms to wild-type-like periodicity (Marrus, 1996)

    The period gene is an internally repetitive gene encoding a tandem array of Thr-Gly codons that are found to be highly polymorphic in length in European natural populations of Drosophila melanogaster. The two major length variants, (Thr-Gly)20 and (Thr-Gly)17, show a highly significant latitudinal cline. The complete sequence of the Thr-Gly region is presented, taken from 91 individuals from 6 natural populations of D. melanogaster: 5 from Europe and 1 from North Africa. These 91 individuals were characterized for polymorphic sites in two other regions, one upstream and one downstream of the Thr-Gly repeat. The haplotypic combinations of Thr-Gly allele were used with flanking markers in an attempt to identify the mechanisms involved in the evolution of the D. melanogaster Thr-Gly region and to infer the phylogenetic relationship existing among the Thr-Gly alleles. There is evidence for both intra- and interallelic mutational mechanisms, including replication slippage, unequal crossing-over, and gene conversion (Rosato, 1996).

    The threonine-glycine (Thr-Gly) encoding repeat within the clock gene period of Drosophila melanogaster is polymorphic in length. The two major variants (Thr-Gly)17 and (Thr-Gly)20 are distributed as a highly significant latitudinal cline in Europe and North Africa. Thr-Gly length variation from both wild-caught and transgenic individuals is related to the flies' ability to maintain a circadian period at different temperatures. This phenomenon provides a selective explanation for the geographical distribution of Thr-Gly lengths and gives a rare glimpse of the interplay between molecular polymorphism, behavior, population biology, and natural selection (Sawyer, 1997).

    Of the mutationally defined rhythm genes in Drosophila melanogaster, period (per) has been the most extensively studied. Three older per mutants, perT, perClk, and per04 have been characterized, along with a novel long-period mutant (perSLIH). Each mutant is the result of a single nucleotide change. perT, perClk, and perSLIH are accounted for by amino acid substitutions; per04 is altered at a splice site acceptor and causes aberrant splicing. perSLIH exhibits a long period of 27 hr in constant darkness and entrains to light/dark (L/D) cycles with a later-than-normal evening peak of locomotion. perSLIH males are more rhythmic than females. perSLIH's clock runs faster at higher temperatures and slower at lower ones, exhibiting a temperature-compensation defect just the opposite of perLong. In the perT mutant, the per-encoded protein (PER) cycles in L/D with an earlier-than-normal peak; this peak in perSLIH is later than normal, and there is a slight difference in the PER timecourse of males vs. females. PER in per04 is undetectable. Two of these mutations, perSLIH and perClk, lie within regions of PER that have not been studied previously and may define important functional domains of this clock protein (Hamblen, 1998).

    Cryptochrome is a major Drosophila photoreceptor dedicated to the resetting of circadian rhythms. How is Cryptochrome mRNA cycling affected by mutations in four clock genes implicated in gene regulation: per, tim, Clock, and cycle? In all single mutants and double mutant combinations, little or no mRNA cycling is found, indicating that cycling requires a functional pacemaker and is not merely light driven. cry mRNA levels are a function of the specific mutant or mutant combination. They are relatively low in the per or tim null mutants as well as in the per;tim double mutant combination, whereas they are relatively high in the Clock and cycle mutants. The double mutants per;Clock and per;cycle also show high cry mRNA levels, indicating an epistatic effect of Clock and cycle over per. Thus, CRY mRNA levels are low in per and tim null mutants, the opposite of what is observed for autoregulation of PER and TIM mRNA levels. CRY mRNA levels are high in clock or cycle mutants, contrary to the low PER and TIM mRNA levels found in these novel clock mutants (Emery 1998 and references).

    The Drosophila lark gene encodes an RNA-binding protein that functions as a regulatory element of the circadian clock output pathway controlling adult eclosion. The lark RNA-binding protein oscillates in abundance during the circadian cycle; importantly, the phasing of the lark rhythm is consistent with gene-dosage studies, which indicate that the protein behaves as a repressor molecule. The lark protein rhythm persists in constant conditions (continuous darkness and constant temperature) and is eliminated by period gene null mutations, confirming that it is under clock control and suggesting that it acts as an output mechanism that mediates the temporal regulation of adult eclosion. lark protein oscillates in abundance within a defined group of neuropeptide (CCAP: seeCardioacceleratory peptide) -containing neurons of the ventral nervous system (VNS), which in other insects are thought to comprise cellular elements of the clock output pathway regulating eclosion (McNeil, 1998).

    The period (per) gene in Drosophila melanogaster provides an integral component of biological rhythmicity and encodes a protein that includes a repetitive threonine-glycine (Thr-Gly) tract, located C-terminal to the PAS domain and several conserved amino acid blocks. Similar repeats are found in the frq and wc2 clock genes of Neurospora crassa and in the mammalian per homologs, but their circadian functions are unknown. In Drosophilids, the length of the Thr-Gly repeat varies widely among species, and sequence comparisons have suggested that the repeat length coevolved with the immediately flanking amino acids. A functional test of the coevolution hypothesis was performed by generating several hybrid per transgenes between Drosophila pseudoobscura and D. melanogaster, whose repetitive regions differ in length by about 150 amino acids. Two per genes, one from D. pseudoobscura and the other from D. melanogaster were selected for analysis; the former has the longest repetitive region of all per genes thus far identified. It consists of about 10 copies of a degenerate Thr-Gly motif to which is added a pentapeptide cassette, which has been derived by slippage from the dipeptide Thr-Gly repeat. There are 30-35 copies of this pentapeptide in different D. pseudoobscura strains, giving the repetitive region a length in excess of 200 amino acids. In comparison, the D. melanogaster repetitive region is composed of about 20 pairs of Thr-Gly, although different strains also have different repeat copy number. The sequences flanking the repeat in blocks H and P, conserved sequences C-terminal to the Pas domain, contain 27 amino acid substitutions between the two parental species, most of which are distributed in block H. Comparing this flanking region to sequences in the databases did not reveal any significant similarities to motifs that might illuminate the function of this region. Transformants carrying per constructs in which the repeat of one species was juxtaposed next to the flanking region of the other (a heterospecific combination) are almost arrhythmic or show a striking temperature sensitivity of the circadian period. In contrast, transgenes in which the repeat and flanking regions are conspecific give wild-type levels of circadian rescue (Peixoto, 1998).

    To learn more about the transgene products, Western blots were performed with head extracts from conspecific and heterospecific transformants over the circadian cycle. Whereas conspecific and wild-type Per produces similar levels of signal, heterospecific Per protein detected was 5-7 times less abundant and similar to the levels of wild-type Per found in tim0 mutants. The conspecific transformants show significant differences in Per levels over the circadian cycle. This temporal profile is similar to that observed with wild-type Per. Wild-type D. melanogaster Per protein also shows a change in mobility and a "broadening" of the Per band over the circadian cycle because of phosphorylation. These effects were present in the wild-type control blots, but the changes in mobility because of phosphorylation appear to be considerably reduced with the conspecific Per combination, even though these transformants gave robust behavioral rhythms. These results support the coevolutionary interpretation of the interspecific sequence changes in this region of the PER molecule and reveal a functional dimension to this process related to the clock's temperature compensation (Peixoto, 1998).

    Requirement of circadian genes for cocaine sensitization in Drosophila

    The circadian clock consists of a feedback loop in which clock genes are rhythmically expressed, giving rise to cycling levels of RNA and proteins. Four of the five circadian genes identified to date influence responsiveness to freebase cocaine in the fruit fly, Drosophila melanogaster. Sensitization to repeated cocaine exposures, a phenomenon also seen in humans and animal models and associated with enhanced drug craving, is eliminated in flies mutant for period, clock, cycle, and doubletime, but not in flies lacking the gene timeless. Flies that do not sensitize owing to lack of these genes do not show the induction of tyrosine decarboxylase normally seen after cocaine exposure. These findings indicate unexpected roles for these genes in regulating cocaine sensitization and indicate that they function as regulators of tyrosine decarboxylase (Andretic, 1999).

    In response to exposure to volatilized freebase cocaine, Drosophila perform a set of reflexive behaviors similar to those observed in vertebrates, including grooming, proboscis extension, and unusual circling locomotor behaviors. Additionally, flies can show sensitization after even a single exposure to cocaine provided that the doses are separated by an interval of 6 to 24 hours. Sensitization, a process in which repeated exposure to low doses of a drug leads to increased severity of responses, has been linked to the addictive process in humans and is potentially involved in the enhanced craving and psychoses that occur after repeated psychostimulant administration. Circadian variation occurs in the agonist responsiveness of Drosophila nerve cord dopamine receptors functionally coupled to locomotor output. This variation is dependent on the normal functioning of the Drosophila period (per) gene, the founding member of the circadian gene family. Because changes in postsynaptic dopamine receptor responsiveness are also seen during cocaine sensitization in vertebrates, flies mutant in circadian functions were examined for alterations in responsiveness to cocaine. Wild-type (WT) flies as well as flies containing a per null mutation, pero, were exposed to 75 µg of cocaine four times over 2 days, and the fraction of flies showing severe responses was quantified after each exposure. Whereas WT flies show sensitization after the initial cocaine exposure, pero flies show no sensitization either to a normal or increased dose even after repeated exposures. As with WT flies, pero flies showed a dose-dependent increase in the severity of responses, and the normal cocaine-induced types of behaviors were observed. In other words, pero flies respond to cocaine but do not sensitize (Andretic, 1999).

    per alleles that either shorten or lengthen the circadian periods show distinct patterns of cocaine responsiveness. The short-period mutants perS and perT both show increased responsiveness to the initial cocaine exposure and weak sensitization to a second 75-µg exposure, with only the sensitization of perS showing statistical significance. Sensitization is not observed in these lines when tested with other cocaine doses. The long-period mutant perL1 showes a normal initial cocaine response but no sensitization to a subsequent exposure (Andretic, 1999).

    Similarly, other circadian genes appear to have effects on cocaine sensitization: Both clock and cycle mutants fail to sensitize when given two doses of cocaine. Because these mutants showed an increased sensitivity to the first exposure, cocaine doses were decreased to 50 µg. The inability of clock and cycle to sensitize is markedly similar to the behavior of pero mutants. The gene product of timeless (tim), Tim, is required for the nuclear translocation of Per and its stability in the cytoplasm; in timo mutants, cytoplasmic Per is degraded and per mRNA levels are constant (Andretic, 1999).

    Recently, a doubletime (dbt) protein with homology to human casein kinase Iepsilon was identified and shown to be required for phosphorylation of Per. Cocaine responses were tested in two viable dbt mutants, dbtS and dbtL, which shorten and lengthen the circadian locomotor period, respectively. dbt mutants require a substantially higher cocaine dose to show behaviors normally observed at 75 µg, but even at these higher doses dbt flies do not show significant sensitization. If the role of dbt in cocaine responsiveness is analogous to its role in circadian behavior, then Per phosphorylation status may be important in regulating both initial cocaine responsiveness and sensitization (Andretic, 1999).

    Modulation of dopamine receptor responsiveness is important in both the sensitization to cocaine in vertebrates and in the circadian modulation of locomotion in Drosophila. To see whether cocaine-sensitized flies would show an increase in the responsiveness of the nerve cord dopamine D2-like receptors, a test was performed using a preparation of behaviorally active decapitated flies that allows direct addition of drugs to the nerve cord. After decapitation, cocaine-sensitized WT flies locomote significantly more than sham-treated controls in response to the dopamine D2-like agonist quinpirole. However, there was no increase in quinpirole responsiveness of pero flies that did not sensitize to repeated cocaine exposures. Thus, similar to the inability of the pero mutant to modulate receptor responsiveness as a function of the time of day, pero is unable to modulate dopamine receptor responsiveness after cocaine exposure. The observation that cocaine sensitization is associated with increased responsiveness of postsynaptic dopamine receptors shows additional similarities between this system and that in higher vertebrates, where a similar relation holds (Andretic, 1999).

    In Drosophila, sensitization requires the trace amine tyramine because the mutant inactive, which is defective in sensitization, shows both reduced tyramine and reduced levels of the enzyme involved in tyramine synthesis, tyrosine decarboxylase (TDC). An active role for tyramine in sensitization is indicated because TDC enzyme activity is induced after a single cocaine exposure, with a time course consistent with that for the development of sensitization. To test if the correlation between induction of TDC activity and behavioral sensitization holds for the circadian mutants, TDC activity was measured in the circadian mutant flies after a single exposure of cocaine. In contrast to WT flies, in which TDC activity is induced after cocaine exposure, the pero, cycle, and clock lines that are defective in sensitization show no such induction; only timo, which shows normal sensitization, induces TDC activity. It thus seems likely that the transcriptional regulator Per, presumably in conjunction with Clock and Cycle, is a direct or indirect regulator of TDC after exposure to cocaine (Andretic, 1999).

    The sensitization defects in inactive and per mutant flies can be distinguished by differences in the ability of tyramine feeding to restore sensitization. The locomotor and cocaine sensitization defects in inactive mutant flies can be rescued by feeding tyramine to adults, but the sensitization defect in pero flies is distinct, because feeding tyramine to pero adults does not rescue sensitization. It is presumed that tyramine from the food can enter tyramine nerve terminals in inactive flies, where it is still subject to a cocaine-stimulated release mechanism that mediates sensitization. The failure of tyramine feeding to rescue sensitization in pero flies is most readily understood if the per gene product is required for this regulated release (Andretic, 1999).

    Similar to inactive , tyramine increases initial cocaine responsiveness in pero flies. Exposure of tyramine-fed pero flies to 35 µg of cocaine induces behaviors normally seen in control flies exposed to 75 µg. Thus, although long-term increase of tyramine levels can affect initial cocaine responsiveness, it is not sufficient for sensitization in flies lacking normal per function (Andretic, 1999).

    A unifying feature of most genes that regulate circadian rhythmicity in Drosophila and vertebrates is the PAS dimerization domain, common to a subset of basic helix-loop-helix transcription factors. Within the circadian cycle, Clock/Cycle heterodimers activate per transcription, whereas Per/Tim heterodimers inhibit the activity of Clock/Cycle. Mutations in per, clock, and cycle share the same cocaine phenotype: a deficiency in the ability to sensitize after one or more drug exposures. This similarity leads to the suspicion that as in circadian behaviors, these genes are functioning in a common pathway. In contrast to the above mentioned genes, the timo mutant showed normal cocaine responses. The implication of this finding is twofold. (1) There must be an as yet unidentified Per binding partner that is specifically involved in regulation of drug responsiveness, and (2) drug responsiveness is likely regulated by per expression in a set of cells distinct from those involved in circadian function. In timo mutants, Per levels are constitutively low; if the same Tim-containing cells were involved in circadian and cocaine responses, timo flies should not sensitize (Andretic, 1999).

    Circadian modulation of dopamine receptor responsiveness in Drosophila melanogaster

    The circadian function of Drosophila dopamine receptors was investigated by using a behaviorally active decapitated preparation that allows for direct application of drugs to the nerve cord. Quinpirole, a D2-like dopamine receptor agonist, induces reflexive locomotion in decapitated flies. The amount of locomotion induced changes as a function of the time of day, with the highest responsiveness to quinpirole during the subjective night. Furthermore, dopamine receptor responsiveness is under circadian control and depends on the normal function of the period gene. The head pacemaker is at least partly dispensable for the circadian modulation of quinpirole-induced locomotion, because changes in agonist responsiveness persist in decapitated flies that are aged for 12 h. This finding suggests a role for the period-dependent molecular oscillators in the body in the modulation of amine receptor responsiveness (Andretic, 2000).

    The circadian variation in locomotor output of the Drosophila nerve cord in response to dopamine agonist stimulation shows two interesting differences from the pattern of locomotor responsiveness in living flies: (1) the rhythms of quinpirole-stimulated nerve cord responsiveness are in opposite phase with in vivo locomotor activity patterns, reaching peak levels during the subjective night, at the time when living flies are least active; (2) there are subtle differences in the activity profiles of the intact and decapitated flies during the light-to-dark transitions (Andretic, 2000).

    The out-of-phase nature of in vivo vs. nerve cord locomotion is most readily explained if the nerve cord responses are modulated as compensatory postsynaptic effects, as has been observed in vertebrates and Drosophila. Postsynaptic dopamine receptors can compensate for differences in the amount of presynaptic release, decreasing sensitivity when dopamine release is high, and increasing when it is low. It has been shown that constitutive overexpression of a stimulatory G-alpha subunit in the dopamine and serotonin neurons, which is expected to increase amine release, results in a decrease in postsynaptic responsiveness to quinpirole. Reciprocally, overexpression of the inhibitory G-alpha subunit or tetanus toxin results in an increased responsiveness to quinpirole. It is speculated that increased dopamine release during the subjective day would stimulate locomotor behaviors, with decreased postsynaptic receptor sensitivity acting as a partially effective compensatory mechanism. At night when dopamine release is presumed to be lower, up-regulation of receptor responsiveness would mediate the enhanced response to quinpirole. Although this model postulates that dopamine release is under circadian control, dopamine synthesis is not, because no variation in brain dopamine content as a function of the 24-h day is found. If modulated dopamine release sets the responsiveness of the quinpirole-sensitive receptors, it could occur even in flies lacking brain input. In invertebrates, the ventral cord, unlike the higher vertebrate spinal cord, contains aminergic cell bodies. Thus, a rhythm of aminergic release under the control of body modulatory circuits could set the responsiveness of quinpirole-sensitive receptors. In intact flies, circadian behavior could be under coordinated control of both the body oscillators and the pacemaker in the brain, because additional dopamine input to the ventral cord originates in the brain and reaches the nerve cord through descending dopamine fibers. Alternatively, the observed circadian modulation of quinpirole sensitivity could be under more direct circadian control. By this scenario, modulation of dopamine receptor sensitivity or other signaling components downstream of the receptor would be modulated, independent of the magnitude of dopamine release (Andretic, 2000).

    These results are most simply consistent with a role for quinpirole-activated dopamine receptors acting in the output pathway from the brain circadian pacemaker. However, none of the results preclude a role for these as of yet unidentified receptors in modulating intercellular responses between cells of the brain circadian pacemaker. Biogenic amines have been implicated in the control of motor behaviors in vertebrates and invertebrates, both in the central and peripheral nervous system. In humans, the importance of dopamine in motor control is most evident in Parkinson's disease, where degeneration of dopamine cell bodies in substantia nigra results in movement disorders. Interestingly, some Parkinson's disease patients display variations in circadian activity patterns, whereas other studies show daily oscillations in the severity of the symptoms, indicating potential communication between the dopamine system and circadian clock. In spinal cats, where the neural axis has been transected, monoaminergic systems are involved in initiation and modulation of locomotion. In arthropod species, injections of dopamine, serotonin, or octopamine into the central nervous system evokes distinct motor postures, suggesting that they are released endogenously to mediate behavior (Andretic, 2000 and references therein).

    Data indicate a role for modulation of dopamine receptor responsiveness in circadian behavior. Modulation of dopamine receptor sensitivity is involved in modulating responses to the indirect amine agonist cocaine, both in vertebrates and in flies. Cocaine functions as a stimulator of reflexive motor and locomotor behaviors both in flies and in vertebrates. It is thus not totally surprising that modulation of responsiveness to cocaine in Drosophila crucially depends on the normal function of a subset of the circadian genes. Given this overlap in functions, it seems likely that there will be altered circadian functions in other mutants showing altered cocaine responses (Andretic, 2000 and references therein).

    Differential regulation of circadian pacemaker output by separate clock genes in Drosophila

    Regulation of the Drosophila pigment-dispersing factor (pdf) gene products was analyzed in wild-type and clock mutants. Mutations in the transcription factors Clock and Cycle severely diminish pdf RNA and neuropeptide (PDF) levels in a single cluster of clock-gene-expressing brain cells, called small ventrolateral neurons (s-LNvs). This clock-gene regulation of specific cells does not operate through an E-box found within pdf regulatory sequences. PDF immunoreactivity exhibits daily cycling, but only within terminals of axons projecting from the s-LNvs. This posttranslational rhythm is eliminated by period or timeless null mutations, which do not affect PDF staining in cell bodies or pdf mRNA levels. Therefore, within these chronobiologically important neurons, separate elements of the central pacemaking machinery regulate pdf or its product in novel and different ways. Coupled with contemporary results showing a pdf-null mutant to be severely defective in its behavioral rhythmicity, the present results reveal PDF as an important circadian mediator whose expression and function are downstream of the clockworks (Park, 2000).

    To assess the effects of clock mutations on pdf expression, the normal cellular distribution of the Drosophila gene's native products were examined. By in situ hybridization, the expression pattern of pdf mRNA has been shown to be similar to that determined with anti-crab-PDH. There are four positive cells in each larval brain hemisphere; these persist into adulthood and become the small ventrolateral neurons (s-LNvs), whose neurites project into a dorsal region of the adult brain. Four large ventrolateral neurons (l-LNvs) also express pdf; these emerge during metamorphosis and send projections into the optic lobe and across the brain midline. Larvae and adults also contain pdf mRNA in the posterior extremity of the CNS (Park, 2000).

    Northern blots reveal no daily rhythm of pdf mRNA abundance, but they could have failed to detect pdf mRNA cycling in a subset of the cells. Thus temporal in situ hybridizations were performed; neither category of pdf-expressing neurons exhibit systematic fluctuations in signal intensities. Therefore, there is no pdf mRNA rhythm for clock mutations to affect (Park, 2000).

    No effect of a period-null mutation on pdf mRNA levels had been detectable in previous Northern blottings. Neither per01 nor a timeless-null mutation affects the RNA's abundance, by Northern blottings and by in situ hybridizations. To search further for regulation by per or tim, adult brains were stained with anti-PDH at different times of day and night. Strikingly, nerve terminals in a dorsal region of the central brain exhibit rhythms of anti-PDH-mediated staining. The neurites that terminate in this region project from the s-LNv cells. In an LD cycle, the peak and trough times for the nerve-terminal cycling are 1 h after lights-on and lights-off, respectively. Staining levels in the perikarya of s-LNvs exhibit some fluctuations but no regular pattern. The adult-specific, larger PDF neurons also exhibit no appreciable cycling of anti-PDH-mediated staining, either in l-LNv cell bodies or in the termini of their neurites that ramify over the surface of the medulla optic lobe (Park, 2000).

    The dorsal-brain, nerve-terminal cycling persists in constant darkness with an ~24-h period in wild type. In that condition the cycle duration is shortened to ~20 h by the perS mutation, which causes behavioral periodicities to be about 5 h shorter than normal. In the dorsal brains of the per01 null mutant, nerve-terminal cycling is abolished, and the signal strengths are very low. However, the immunohistochemical procedure performed on these brain sections is not very sensitive. Therefore, a quantitative fluorescence method was used, the better to judge PDF staining intensities in whole-mounted brains. At the peak and trough time-points, nerve-terminal signals in wild type are again higher in the early morning compared with the early night. This temporal difference is not observed in the dorsal brains of the arrhythmic per01 and tim01 mutants. In per01, the staining intensities at both times are nearly identical and at levels intermediate between the per+ peaks and troughs. In tim01, the PDF terminal signals are also the same at the two time-points but significantly higher than in tim+ and. The mutational effects of these clock genes on daily fluctuations of PDF abundance at certain nerve terminals indicate that an aspect of this peptide's regulation is, in one way clock controlled, and in another was posttranslationally regulated (Park, 2000).

    Specific genetic interference with behavioral rhythms in Drosophila by expression of inverted repeats

    A new experimental technique is described that allows for a tissue-specific reduction of gene activity in the Drosophila nervous system. On the basis of the observation that certain gene functions can be ubiquitously blocked by injecting double-stranded RNA into Drosophila embryos, a method was employed to permanently interfere with an individual gene function in a predetermined cell type. This was achieved by the formation of an inverted-repeat RNA sequence in the tissue of interest under control of the GAL4/UAS binary expression system. As an example, inverted-repeat-mediated interference with the period gene produces a hypomorphic period phenotype. A selective decrease of Period RNA appears to be a component of the cellular response (Martinek, 2000).

    Sequences from two different regions of the period open reading frame were used for the generation of inverted repeats. perCt contains a carboxy terminal fragment without any known homologies. perPAS contains a sequence encoding a putative protein dimerization domain, the PAS domain. The length of a single repeat in both cases is close to 1 kb. The location of the inverted-repeat sequence and the length of the gap between the repeats of 67 bp were chosen to facilitate cloning (Martinek, 2000).

    Inverted repeats were expressed using the GAL4/UAS binary expression system in cells relevant for rhythmic locomotor activity. To this end, a transgenic line that expresses GAL4 under the control of the timeless promoter, timeless-GAL4, was employed. The timeless gene is another essential clock component and therefore is expressed in all cells with a functioning clock. These cells include the lateral neurons (LNs), where the regulator of rhythmic locomotor activity is believed to be located. timeless-GAL4 is shown to be active in cells expressing timeless (Martinek, 2000).

    Expression of perCt-inverted repeat (perCt-IR) and perPAS-inverted repeat (perPAS-IR) results in a lengthening of the average period of the circadian locomotor activity cycle by ~2 hr compared to timeless-GAL4/+. Almost identical phenotypes have been observed for several independent transgenic lines. The phenotype is nearly fully penetrant. Only 10% of the flies expressing perCt-IR and only 3% of the flies expressing perPAS-IR showed a period of <25 hr compared to 9% of wild-type controls with periods of >24 hr. Since there is an inverse correlation between period dosage and period length, the described phenotype is in good agreement with a decrease in Period RNA levels caused by expression of the inverted repeats. Very long period rhythms of >35 hr can be caused by an extremely strong reduction of Period RNA abundance (20-fold). However, in this study the maximal increase in period length was 3 hr. This suggests that these experiments reduce, but do not eliminate, expression of period (Martinek, 2000).

    A Timeless-independent function for Period proteins in the Drosophila clock

    Circadian (24 hour) Period (Per) protein oscillation is dependent on the double-time (dbt) gene, a casein kinase Iepsilon homolog. Without dbt activity, hypophosphorylated Per proteins over-accumulate, indicating that dbt is required for Per phosphorylation and turnover. There is evidence of a similar role for casein kinase Iepsilon in the mammalian circadian clock. A new dbt allele, dbtar, has been isolated that causes arrhythmic locomotor activity in homozygous viable adults, as well as molecular arrhythmicity, with constitutively high levels of Per proteins, and low levels of Timeless (Tim) proteins. Short-period mutations of per, but not of tim, restore rhythmicity to dbtar flies. This suppression is accompanied by a restoration of Per protein oscillations. These results suggest that short-period per mutations, and mutations of dbt, affect the same molecular step that controls nuclear Per turnover. It is concluded that, in wild-type flies, the previously defined Per 'short domain' may regulate the activity of DBT on Per (Rothenfluh, 2000b).

    In a screen for mutations affecting Drosophila locomotor activity rhythms, a new dbt allele, dbtar, was recovered that results in arrhythmic flies when homozygous. Neither the strongly hypomorphic dbtP allele, nor a deficiency, Df(3R)tll-g, which removes the dbt gene, are able to complement the new mutation. At the molecular level, dbtar stops the oscillation of Per and Tim proteins, and causes under-accumulation of Tim, and over-accumulation of Per. These phenotypes are similar to those of dbtP mutants, suggesting that dbtar is a reduced-function allele. However, the degree of phosphorylation of constitutively produced Per in dbtar ranges from hypo- to hyperphosphorylated, whereas over-accumulating Per is hypophosphorylated in dbtP. dbtar flies therefore retain a higher level of DBT-mediated kinase activity than dbtP. The fact that ~70% of dbtar homozygotes are viable, compared with the complete pupal lethality of dbtP and dbtdco alleles also supports higher kinase activity of DBTAR. The single missense mutation associated with dbtar results in a His126 to Tyr mutation. His126 is highly conserved in casein kinase Iepsilon family members, but Tyr126 is found in casein kinase Igamma. The specificity of DbtAR may, therefore, be altered, resulting in some, but not sufficient, Per phosphorylation for turnover (Rothenfluh, 2000b).

    dbtar shows some residual, weak, long-period rhythms, when heterozygous with the dbtP allele or Df(3R)tll-g. To see if the frequency of rhythmicity could be increased, dbtar was crossed into various short-period mutant backgrounds. The two short-period per alleles, perT (16 hours), and perS (19 hours), substantially reduce the frequency of arrhythmia: dbtar/dbtP flies are rescued to almost complete rhythmicity by perT, perS, and even perT/+. Rescue of dbtar/Df(3R)tll-g is less pronounced, but the ~90% arrhythmicity observed in this genotype is suppressed by perT to over 50% rhythmicity. The suppression of arrhythmia is also accompanied by an increased strength of the rhythms. This is the first report of a rescue of genetically determined arrhythmia by a second-site mutation (Rothenfluh, 2000b).

    Unlike the 20.5 hour heterozygous perT background, the 20.5 hour timS1 background is not able to rescue rhythms in dbtar/dbtP and dbtar/Df(3R)tll-g heterozygotes. There is, therefore, a specific genetic interaction between short-period per alleles and dbtar, and dbtar is not generally rescued toward rhythmicity by any period-shortening allele. Specificity of this rescue is also indicated by the finding that a 28-hour period per allele, perSLIH, does not suppress dbtar arrhythmia. Furthermore, rescue of arrhythmicity is also specific to dbtar, because perT heterozygotes do not suppress the arrhythmia from the tim01 mutation (Rothenfluh, 2000b).

    How can this allele-specific suppression of dbtar arrhythmia be explained? In perS flies, PerS disappears prematurely from nuclei , and Per from perS and perT flies falls to trough levels prematurely at the end of the night. Evidently per-short mutations increase the turnover of nuclear Per proteins. Because dbt affects the stability of cytoplasmic and nuclear Per, dbtar and per-short alleles may both affect nuclear Per stability, although in opposite directions. PerS and PerT might be turned over in the nucleus despite reduced Dbt activity in dbtar flies, thus completing the molecular cycle by terminating Per auto-repression, and resulting in molecular and behavioral rhythms. Conversely, in per+;dbtar flies, the program of nuclear Per protein degradation may be obstructed, resulting in continued repression of per and tim. Altered regulation of this sort should be reflected in the Tim under-accumulation observed in dbtar homozygotes (Rothenfluh, 2000b).

    To test whether the behavioral rescue is associated with restoration of molecular oscillations, Per and Tim protein time courses were examined on Western blots from perT;dbtar/dbtP flies. Per and Tim levels oscillate, and Per reaches trough levels at ZT 10 (in LD), and CT 18 (first day in DD). In this time span progressive phosphorylation of Per is also observed, even though the transition from hypo- to hyper-phosphorylation takes ~20 hours, compared to 8-12 hours in wild-type flies. This reduced rate of phosphorylation is probably a result of the dbtar mutation, and might explain the 31-hour behavioral period of perT;dbtar/dbtP flies (Rothenfluh, 2000b).

    Normally, lights-on in the morning results in Tim degradation, followed by progressive phosphorylation and degradation of hyperphosphorylated Per in 5-7hours. In the presence of light, hyperphosphorylated forms of Per are found that are not eliminated from dbtar/dbtP flies. In perT;dbtar/dbtP flies, however, Per levels are reduced by exposure to light and hyperphosphorylated Per proteins are lost, indicating that PerT proteins are more easily degraded than wild-type Per proteins when DBT activity is compromised. Since progressive phosphorylation of PerT is observed in perT;dbtar/dbtP flies, and PerT proteins turned over following exposure to light in an LD cycle are hyperphosphorylated, Per proteins should still require phosphorylation for degradation in this background. A hypophosphorylated form of PerT was found in the presence of light in these flies that might be freshly synthesized cytoplasmic Per. The dbtP mutation results in stable, hypophosphorylated cytoplasmic Per, and similarly, dbtar may also increase Per's cytoplasmic stability (Rothenfluh, 2000b).

    To test whether the increased degradation of PerT proteins occurs in nuclei of perT;dbtar/dbtP flies, head sections were stained for Per protein. There is a pronounced diminution of nuclear Per from ZT 2-10 in perT;dbtar/dbtP photoreceptors, whereas similar levels of Per are observed at these two time points in dbtar and in dbtar/dbtP photoreceptors. It is concluded that increased nuclear turnover of PerT allows completion of the molecular cycle in the presence of the Per-stabilizing mutation dbtar (Rothenfluh, 2000b).

    The specific molecular mechanism affected by the interaction of short period mutations of per and dbtar is unknown. However, earlier work has established that the perS mutation maps to a ~30 amino acid domain of Per in which most amino acid substitutions produce similar short-period phenotypes. A simple deletion of 17 amino acids {SERDSVMLGEISPHDDY} from this Per 'short domain' also reduces period-length as in perS, and the perT mutation has been mapped to this interval of Per. Together the results indicate that in wild-type flies, Per's short domain somehow inhibits the action of DBT. Because DBT and Per physically bind to each other in vitro, in cultured Drosophila cells, and in vivo, the short domain might influence a pattern of physical association between Per and DBT that retards Per phosphorylation in wild-type flies. Alternatively, because Per is repeatedly phosphorylated in vivo, and hyperphosphorylation appears to be required for Per degradation, the short domain may influence a temporal sequence of Per phosphorylation. Additional evidence that per-short mutations and dbt affect the same step in the cycle comes from the finding that per-short and dbt-short double mutant combinations show an unusual non-additive phenotype (Rothenfluh, 2000b).

    Circadian rhythms of female mating activity governed by clock genes in Drosophila

    Wild-type Drosophila melanogaster displays a robust circadian rhythm in the mating activity, and these rhythms are abolished in period- or timeless-null mutant flies (per01 and tim01). Circadian rhythms are lost when rhythm mutant females are paired with wild-type males, demonstrating that female mating activity is governed by clock genes. Furthermore, an antiphasic relationship in the circadian rhythms of mating activity was detected between D. melanogaster and its sibling species Drosophila simulans. Female- and species-specific circadian rhythms in the mating activity of Drosophila seem to cause reproductive isolation (Sakai, 2001).

    To determine whether mating activities differ between day and night, the mating frequencies of D. melanogaster maintained in 12:12 LD cycles were measured. The mating activities of 3-day-old flies do not significantly differ between day (ZT3) and night (ZT12). However, the mating activities of 5-, 7-, and 9-day-old flies significantly differ between day and night (Sakai, 2001).

    To determine whether the mating activity of D. melanogaster fluctuates over the day, the mean mating frequencies of two strains (Canton-S and OGS-4) at 9 days of age were measured. The mating activities of both D. melanogaster strains are similar over the day under LD cycles (lights on at 9:00 and lights off at 21:00). The mating activities of both strains at ZT12 are significantly lower than at other times. The rhythms of 7-day-old Canton-S flies were the same under the same LD conditions. Furthermore, the rhythms of 7-day-old flies that are kept under different LD cycles (lights on at 6:00 and lights off at 18:00) after eclosion are similar to those of 7- and 9-day-old flies kept under LD cycles with lights on at 9:00. These results indicate that the rhythms of Drosophila mating activity become synchronized (entrained) to daily LD cycles. To determine whether these rhythms are controlled by an endogenous clock, the mating activities of flies on day 2 of DD after 7 days of entrainment in LD cycles were measured. The reduction of mating activity at circadian time (CT) 12 remains intact in both strains under DD as well as in LD. These results indicate that the mating activity of D. melanogaster is under the restricted control of an endogenous clock (Sakai, 2001).

    To know whether the mating-activity rhythms of Drosophila are controlled by circadian clock genes, the mating activity was measured in per01and tim01 flies that lack rhythms in adult emergence and locomotor activity. In contrast to the two wild-type strains under LD cycles, these mutant flies do not recover mating activity within 3 to 6 h from lights off. When the flies are allowed to mate for 15 min, mating activities in these rhythm mutants are not reduced at CT12 in DD. When per01 flies are allowed to mate for 25 min, mating activity over the day is high (37%-50%) and not reduced at CT12. These results indicate that the circadian clock genes, per and tim, affect the circadian rhythm of Drosophila mating activity. Mating activities of the per01 and tim01 mutants are elevated in the morning. However, mating activity is not elevated in the two mutants under DD. These results indicate that light signals also directly affect mating activity in the morning (Sakai, 2001).

    In D. melanogaster, the specific neurons of the optic lobe seem to play a major role as pacemakers for locomotor activity rhythms, because a transgenic line in which per expression is restricted to the lateral neurons has rhythmic locomotor activity. Further evidence is provided by studies of disconnected (disco) mutants that have a severe defect in the optic lobe and are missing lateral neurons. Both locomotor activity and eclosion of the disco mutant are arrhythmic under DD. The present study found that mating activities of disco mutants, like those of per01 and tim01 mutants, are not reduced at CT12. Taken together, these results suggest that arrhythmicity in the mating activities of disco mutants is also caused by defective lateral neurons (Sakai, 2001).

    The specific role of sex that may be involved in the circadian rhythm of mating activity can be investigated in rhythm mutants. To determine whether the robust circadian rhythm in mating activity shown in the wild-type is caused specifically by males, females, or a combination of both sexes, Canton-S females were paired with either tim01 or Canton-S males, and tim01 females were paired with either tim01 or Canton-S males. Mating-activity rhythm was abolished in tim01 females crossed with Canton-S males. In contrast, mating-activity rhythm was undetectable regardless of which females were mated with tim01 males. The mating activity of such pairs was very low over the day, suggesting that the tim gene or the genetic background of the tim01 mutant is responsible for low courtship activity of the mutant males. Other experiments demonstrate that females are responsible for generating the circadian rhythm of mating activity in Drosophila. The findings suggested that females need lateral neurons to generate these rhythms and that a female-specific circadian clock suppresses mating activity at CT12 (Sakai, 2001).

    Significant differences exist in the mating activities of 5-, 7-, and 9-day-old flies between day and night. To determine whether these differences are caused specifically by males, females, or both sexes, 9- and 3-day-old males were paired with 3- and 9-day-old females, respectively. When males are paired with 9-day-old females, day/night activities clearly differ. In contrast, these differences are absent when 3-day-old females are paired with 3- and 9-day-old males. Thus, Drosophila females contribute to the day/night differences in the mating activities; the experiments suggest that a female-specific circadian clock drives mating activities at least after 5 days of age. In 3-day-old females, the mechanisms to modulate the mating activity may be undeveloped. Alternatively, some sort of mating drive may overwhelm clock control in the youngest females (Sakai, 2001).

    To know whether the per gene affects the reduction of mating activities at CT12, the mating activity in combinations of both types of hsp-per females and Canton-S males was examined. No differences in the mating activities between CT12 and CT18 were detected in the HS- experiment as well as in the per01 mutant. However, mating activities at CT12 are significantly lower than those at CT18 in the HS+ experiment. These results are similar to those from the wild-type, suggesting that per gene expression causes the reduction in mating activity at CT12, and that arrhythmicity in the mating activity of the per01 mutant is caused by the per mutation of female flies rather than by the genetic background of the mutants (Sakai, 2001).

    The results of the present study demonstrate that mating activity is driven by two mechanisms in Drosophila. One is a circadian pacemaker consisting of clock genes and the other is the direct effect of light. The mating-activity rhythm of D. melanogaster females is under the restricted control of circadian clock genes, and the lateral neurons might be essential to generate the rhythm. Flies, especially males, use olfactory cues for mating, and the circadian rhythm of the olfactory response is robust in Drosophila . Olfactory responses of the wild-type are elevated in the middle of the night in LD cycles, but mating activities are decreased during the early part of the night. Furthermore, the lateral neurons are insufficient to sustain olfactory rhythm but the optic lobe, including the lateral neurons, seems to be essential for mating-activity rhythm according to these results. Thus, the mechanism that generates the mating-activity rhythms might be independent of that which generates olfactory rhythms. A female sex pheromone attracts male courtship in Drosophila, and the sound produced by male wing vibration, referred to as courtship song, affects female receptivity. One explanation for the generation of female mating activity in Drosophila is that females show circadian rhythms in pheromone release and/or responses to auditory signals (Sakai, 2001).

    Loss of circadian clock function decreases reproductive fitness in males of Drosophila

    Circadian coordination of life functions is believed to contribute to an organism's fitness; however, such contributions have not been convincingly demonstrated in any animal. The most significant measure of fitness is the reproductive output of the individual and species. The consequences of loss of clock function on reproductive fitness in Drosophila have been examined with mutated period (per0), timeless (tim0), cycle (cyc0), and Clock (ClkJrk) genes. Single mating among couples with clock-deficient phenotypes results in ~40% fewer progeny compared with wild-type flies, because of a decreased number of eggs laid and a greater rate of unfertilized eggs. Male contribution to this phenotype was demonstrated by a decrease in reproductive capacity among per0 and tim0 males mated with wild-type females. The important role of clock genes for reproductive fitness was confirmed by reversal of the low-fertility phenotype in flies with rescued per or tim function. Males lacking a functional clock show a significant decline in the quantity of sperm released from the testes to seminal vesicles, and these tissues displayed rhythmic and autonomous expression of clock genes. By combining molecular and physiological approaches, a circadian clock was identified in the reproductive system and its role in the sperm release that promotes reproductive fitness in D. melanogaster was defined (Beaver, 2002).

    To determine whether the low-sperm phenotype is correlated with clock function in the reproductive system, the activity of clock genes in these tissues was studied. Spatial expression of per and tim was evaluated in flies that carry a per-lacZ reporter construct or express green fluorescent protein under control of the tim promoter. Both reporters exhibited strong activity in the lower testes and SVs, weak activity in the ejaculatory duct and the upper testes, and no activity in the paragonial (accessory) glands. Immunocytochemical analysis shows rhythmic expression of PER and TIM proteins limited to the lower testes and the SVs. PER and TIM were not detected at ZT 8, whereas both proteins are ubiquitously expressed in the nuclei of the epithelial cells forming the lower testes and the SV at ZT 20. PER and TIM proteins are absent in per01, per04, and tim01 flies. However, similar to wild-type, distribution of both proteins in the SV and lower testes was observed in the reproductive system of per01 mutants rescued with a per+ construct and tim01 mutants transformed with a tim+ construct. Nuclear localization of PER and TIM was evident in those flies at ZT 20 (Beaver, 2002).

    Because the functional clock of the fruit fly involves out-of-phase cycling of per and Clk mRNAs, in situ hybridization of the male reproductive system was performed with antisense probes for both genes. Both per and Clk mRNA were detected in the lower-testes-SV epithelium. The level of per mRNA was low at ZT 4 and high at ZT 16, whereas Clk mRNA shows cycling in the opposite phase with high levels at ZT 4 and low levels at ZT 16. Taken together, these results demonstrate cycling of clock components that is similar to patterns observed in fly brains and is consistent with the existence of a circadian clock in the male reproductive system (Beaver, 2002).

    To elucidate the autonomy of the testes-SV circadian system, BG-luc (reporting per) or tim-luc transgenic flies were used. Testes-SV complexes were dissected from these flies and individually cultured in vitro in LD followed by dark/dark (DD) cycles and a return to LD. Isolated organs show clear, high-amplitude cycling of BG-luc and tim-luc activity during the initial LD cycles with peak expression during the night. Quantitative analysis of the data reveals that 59% of testes-SVs from BG-luc flies and 76% of same organs from tim-luc flies are rhythmic in vitro in the circadian range. On transfer to DD, cycling continues in 36% of both BG-luc and tim-luc organs with reduced amplitude. When the free-running cultures are returned to LD cycles, the amplitude increased for both constructs, demonstrating direct light responsiveness of the testes-SV circadian system. When both constructs were crossed into genetic backgrounds carrying a loss-of-function mutation for the respective clock gene (BG-luc into per01 and tim-luc into tim01), circadian oscillations were eliminated, indicating that wild-type alleles of the two clock genes are needed to support reporter-gene cycling (Beaver, 2002).

    Stress response genes protect against lethal effects of sleep deprivation in Drosophila

    Sleep is controlled by two processes: a homeostatic drive that increases during waking and dissipates during sleep, and a circadian pacemaker that controls its timing. Although these two systems can operate independently, recent studies indicate a more intimate relationship. To study the interaction between homeostatic and circadian processes in Drosophila, homeostasis was examined in the canonical loss-of-function clock mutants period (per01), timeless (tim01), clock (Clkjrk) and cycle (cyc01). cyc01 mutants show a disproportionately large sleep rebound and die after 10 hours of sleep deprivation, although they are more resistant than other clock mutants to various stressors. Unlike other clock mutants, cyc01 flies show a reduced expression of heat-shock genes after sleep loss. However, activating heat-shock genes before sleep deprivation rescues cyc01 flies from its lethal effects. Consistent with the protective effect of heat-shock genes, is the observation that flies carrying a mutation for the heat-shock protein Hsp83 (Hsp8308445) show exaggerated homeostatic response and die after sleep deprivation. These data represent the first step in identifying the molecular mechanisms that constitute the sleep homeostat (Shaw, 2002).

    A sleep-like state has been described in Drosophila on the basis of its similarities to mammalian sleep. This state is characterized by increased arousal thresholds and is regulated homeostatically. Like mammalian sleep, it is abundant in young flies, decreases in older animals and is modulated by stimulants and hypnotics. Perhaps the most important similarity between mammals and flies is homeostatic regulation: when flies are kept awake, they show a large compensatory increase in sleep the next day (Shaw, 2002).

    In mammals, the circadian pacemaker alternately promotes and maintains both wakefulness and sleep. Although the circadian pacemaker and the sleep homeostat can interact, little is known about the underlying mechanisms. To evaluate this relationship, homeostasis was evaluated in clock mutants maintained in constant darkness (DD) and deprived of sleep for 3, 6, 9 and 12 h. Under these conditions, sleep is evenly distributed across the day. Upon release from sleep deprivation, wild-type Canton-S flies recover ~30%-40% of the sleep that they lost within 12 h. per01 and Clkjrk show a more prominent sleep rebound, reclaiming ~100% of lost sleep within 12 h. tim01 flies did not show a homeostatic response after 3–6 h of sleep deprivation but displayed a sleep rebound similar to that of per01 and Clkjrk flies after 7, 9 and 12 h of sleep deprivation (Shaw, 2002).

    To determine whether death in cyc01 flies is due specifically to sleep deprivation or to hypersensitivity to any environmental challenge, per01, tim01, Clkjrk, cyc01 and Canton-S flies were subjected to several stressors including heat stress, oxidative stress, starvation, desiccation and physical stress. cyc01 flies were as sensitive, but no more so than other genotypes to desiccation and vortex-mixing and survived longer than per01, tim01 and Clkjrk flies when challenged with heat, oxidative stress and starvation. Canton-S flies, which have an intact clock, were more resistant to starvation and desiccation than tim01, Clkjrk and cyc01 flies. These data indicate that cyc01 mutants are vulnerable to prolonged wakefulness in itself and are not merely hypersensitive to non-related stressors (Shaw, 2002).

    Temporal mating isolation driven by genetic variation in period

    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. 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. Transgenic flies carrying the natural per genes from these two Drosophila species have been used 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 (Tauber, 2003).

    The daily locomotor activity profiles of drosophilids are under the control of the circadian system, and the sex-linked clock gene period (per) accounts for the species-specific variation observed in activity patterns between D. melanogaster and D. pseudoobscura. A recent study has demonstrated that mating activity is also under the control of the circadian system, and that different sympatric species maintain different mating schedules. As transient locomotor activity levels have been observed to correlate with mating activity, it was of interest to ask whether these two species might have different mating rhythms that might also be per controlled. Initially the free-running activity of D. melanogaster and D. pseudoobscura males in constant darkness (DD) at different temperatures was reexamined; the previously reported species-specific profiles were confirmed and extended. D. melanogaster is slightly bimodal in its locomotor activity, illustrated by a major peak early in the subjective day, while D. pseudoobscura has its major peak around subjective dusk. Transformant lines carrying the hemizygous D. melanogaster per transgene on a per01 background (mel) rescue rhythmicity with the same melanogaster pattern, whereas per01 transformants similarly hemizygous for the D. pseudoobscura per transgene, mps1, show the pseudoobscura pattern, illustrated by a peak of activity later in the subjective day. Because the free-running period of mps1 transformants on a per01 background is long (>28 hr) and, even in rhythmic individuals, the strength of the cycle is poor, the mps1 transgene was crossed into a wild-type per+ background and the insert was made homozygous (per+/per+/Y; mps1/mps1). These flies have a robust free-running period of ~24 hr (24.3 hr for both males and females), and, in spite of having multiple doses of per, the locomotor patterns are clearly pseudoobscura like and have a prominent peak in the late subjective day, indicating the dominance of the mps1 transgene over the endogenous melanogaster per+ gene (Tauber, 2003).

    The results have extended the original observation that per controls species-specific behavioral instructions and have revealed that, irrespective of the relative dosage of the heterospecific per (trans)gene, the species-specific switch from melanogaster-like to pseudoobscura-like locomotor behavior is dominant and fully penetrant. Similar experiments have identified the per gene as determining species specificity of the courtship song rhythm between the sibling species D. melanogaster and D. simulans. The fact that species differences in complex behaviors of adaptive importance can be controlled so tightly by a single locus adds further support to the emerging view that the 'infinite' view of the genetic basis of adaptations, by which small genetic differences accumulate over many generations at many loci, may not represent a general phenomenon (Tauber, 2003).

    D. melanogaster and D. pseudoobscura coexist in sympatry, and although heterospecific matings have not been reported, and indeed are unlikely to occur, it has been noted that, within their shared natural environment, the latter species shows a peak of mating behavior just before darkness, whereas D. melanogaster mating behavior is more frequent in the hours before dusk. Thus, the mating rhythm data experimentally confirm these field observations. Locomotor activity is also expected to affect mating; for example, males are more attracted to moving females. However, even though species-specific cycles of mating activity initially appeared to correlate nicely with those of locomotor activity, the sexual rhythm is not in phase with the locomotor rhythm and lags by several hours in both D. melanogaster and D. pseudoobscura and their corresponding transformants. This would suggest that periods of high locomotor activity are associated more closely with other functions, such as foraging, rather than mating. Thus, the initial hypothesis that drove this study, namely, that periods of active locomotor behavior might be causally related to enhanced mating behavior, was not supported. Nevertheless, these two behavioral rhythms may be a manifestation of the same central oscillator, so a comparative molecular analysis of the per mRNA and protein cycles in the two species would be of obvious interest (Tauber, 2003).

    In the assortative mating experiments, the interspecific per transformants all have natural 24-hr circadian periods, allowing simulation of how different natural alleles of per might drive pre-mating isolation. In addition, by using wingless males, any effects of the different species per genes on the courtship song cycles of the transformant hosts are bypassed. The results reveal changes in per-mediated assortative mating throughout the circadian cycle. Assortative mating is generally enhanced relative to controls at all circadian phases when the two strains carry different species per transgenes, but it is particularly prominent at dusk. The D. pseudoobscura mating rhythm reaches a peak very late in the subjective day and is similar to that of D. simulans. Since D. melanogaster and D. simulans are sympatric sibling species, one can extrapolate from the data (for D. pseudoobscura, read D. simulans) that the time-dependent peak in assortative mating would be activated when the greatest risk of heterospecific mating is approaching, i.e., between CT9 and CT12 for D. melanogaster and D. simulans. This might explain why control transformants that have different genetic backgrounds, but the same melanogaster per transgene, show some level of assortative mating at CT12. It can thus be imagined that this dusk-related effect is considerably amplified when flies are also present that carry a per transgene from another species that may alter the phasing of any mate choice cycle (Tauber, 2003).

    per was originally considered as a 'speciation gene', based on its role in modulating the interpulse interval of the male courtship song, which is a highly species-specific song character. Behavioral genes that affect sexual communication are obvious candidates for reproductive isolation. However, such loci need to evolve simultaneously in the systems controlling signal production (usually in the male) and signal recognition (in the female). Mechanisms for genetic coupling in Drosophila between sender and receiver have been suggested, for example, based on genes that encode mechanosensors, which in turn may control sensory feedback. In the case of per, genetic variations that alter the male song character do not make the females carrying these mutations respond preferentially to the male mutant song. In the present study a simpler mechanism is proposed by which per may act as a putative speciation gene merely by shifting the daily timing of mating behavior. per is the first identified single gene in Drosophila that alters assortative mating. This might occur by per conveying species-specific phase differences in sensitivity rhythms within sensory pathways, such as olfaction. Indeed, such cycles have been documented from chemosensory cells in the antennae, which generate circadian physiological rhythms in response to odorants. Thus, a genetic analysis of per-mediated assortative mating with olfactory variants may provide an initial dissection of the neural mechanisms that generate assortative mating in this genus (Tauber, 2003).

    There are currently no strong candidates for female preference genes. Segments of chromosomes have been identified in the D. melanogaster Zimbabwe population, and these chromosomes may be responsible for the strong isolation of this race from the worldwide form of D. melanogaster. Similarly, part of the sexual isolation between D. pallisoda and D. ananassae maps near the Delta locus. Under laboratory conditions, natural species-specific variation at a single locus, per, can potentially lead to temporal mating isolation within a single species, via the effects of changes in mating rhythms. The implications of such a direct relationship between gene sequences and mate choice, whatever the intervening physiological mechanisms, may have important implications for the processes of sympatric speciation (Tauber, 2003).

    Regulation of Copulation Duration by period and timeless in Drosophila

    The circadian clock involves several clock genes encoding interacting transcriptional regulators. Mutations in the Drosophila clock genes period, timeless, Clock and cycle produce multiple phenotypes associated with physiology, behavior, development, and morphology. It is not clear whether these genes always work as clock components or may also act in some unknown pleiotropic fashion. per and tim are shown to be involved in a novel, male-specific phenotype that affects behavioral timing on the order of minutes. Males lacking per or tim copulate significantly longer than males with normal per or tim function, while females do not show this effect. No correlation between fertility and extended copulation duration was found. Several lines of evidence suggest that the time in copula (TIC) is not regulated by the known clock mechanism: (1) the period of free-running clock oscillations does not appear to affect this phenotype; (2) constant light, which abolishes the clock function, does not alter TIC (3) mutations in the positively acting clock transcription factors, Clk and cyc, do not affect TIC. This study extends the repertoire of behavioral functions involving per and tim genes and uncovers another time scale over which these genes may act (Beaver, 2004).

    The genetic basis for copulation duration in Drosophila was first demonstrated through the directional selection of flies for short and long copulation durations over subsequent generations. Since that time, only a handful of genes have been identified that affect copulation duration. Most of these genes appear to affect the development of physical structures or neuronal circuits necessary for successful copulation. Consequently, male flies may display difficulties with the physical interaction of copulation, such as withdrawing genitalia and dismounting from females. In contrast, males observed in this study had no apparent defects of this kind; they terminated prolonged copulations in a manner similar to wild-type males. This suggests that per and tim are somehow involved in measuring the duration of mating behavior as part of their broad functions related to the timing of biological processes on different time scales ranging from seconds to days (Beaver, 2004).

    There are two possible mechanisms by which per and tim could participate in determining copulation duration. (1) per and tim may exert pleiotropic effects related to their involvement in development of the fly. It is known that per and tim are expressed during embryonic, larval, and pupal stages; therefore, these genes could affect the development of the CNS, peripheral sense organs, and muscles, leading to altered behavior in adults. (2) per and tim may regulate copulation duration via their expression in sexually mature males. These genes are expressed in the male reproductive system as key components of peripheral circadian oscillators. However, this clock-related expression is not likely to contribute to the extended TIC phenotype because TIM expressed in the male reproductive organs is light sensitive, and constant light does not produce an extended TIC phenotype. These results suggest that the TIC phenotype may involve tissues in adult males where per and tim are expressed in constant light and are not regulated by cyc and Clk. Such unorthodox behavior of per and tim has in fact been reported in several studies. For example, expression of TIM and PER in the fly ovary persists in constant light and does not depend on cyc and Clk; a similar situation could conceivably occur in some as yet unidentified male peripheral tissues. With regard to the nervous system, it has been shown that certain subsets of larval and adult brain neurons show high levels of Tim and Per during the light phase of an LD cycle. Moreover, both proteins were detected in certain brain neurons in cyc and Clk loss-of-function mutants. Such putative neural sites where levels of Tim and Per would be light insensitive and independent of Clk and cyc function may be involved in regulating duration of copulation (Beaver, 2004).

    Previous studies have shown that per and tim play significant roles in fly reproductive fitness as regulators of fertility in both male and female flies. This current study further extends the range of per and tim functions in reproduction by demonstrating their interesting role as key modulators of an important reproductive behavior (Beaver, 2004).

    Disruption of Cryptochrome partially restores circadian rhythmicity to the arrhythmic period mutant of Drosophila

    The Drosophila circadian clock is generated by interlocked feedback loops, and null mutations in core genes such as period and timeless generate behavioral arrhythmicity in constant darkness. In light-dark cycles, the elevation in locomotor activity that usually anticipates the light on or off signals is severely compromised in these mutants. Light transduction pathways mediated by the rhodopsins and the dedicated circadian blue light photoreceptor cryptochrome are also critical in providing the circadian clock with entraining light signals from the environment. The cryb mutation reduces the light sensitivity of the fly's clock, yet locomotor activity rhythms in constant darkness or light-dark cycles are relatively normal, because the rhodopsins compensate for the lack of cryptochrome function. Remarkably, when a period-null mutation was combined with cryb, circadian rhythmicity in locomotor behavior in light-dark cycles was restored, as measured by a number of different criteria. This effect was significantly reduced in timeless-null mutant backgrounds. Circadian rhythmicity in constant darkness was not restored, and Tim protein did not exhibit oscillations in level or localize to the nuclei of brain neurons known to be essential for circadian locomotor activity. Therefore, this study uncovered residual rhythmicity in the absence of period gene function that may be mediated by a previously undescribed period-independent role for timeless in the Drosophila circadian pacemaker. Although a molecular correlate for these apparently iconoclastic observations is not available, a systems explanation for these results is provided, based on differential sensitivities of subsets of circadian pacemaker neurons to light (Collins, 2005).

    This study has revealed a surprising and intriguing restoration of circadian rhythmicity in LD cycles in per01; cryb flies. This partial rescue can even be extended to the adaptive thermal change in locomotor behavior mediated by 3' UTR splicing of the per transcript (Collins, 2004: Majercak, 2004; Majercak, 1997). A number of criteria have been used to dissect rhythmic behavior, including phase shifting in response to light pulses in LD and the use of T cycles to suggest that a residual oscillation, rather than an hourglass, underlies the behavior of the double mutant. The phase shifting of the per01; cryb oscillator is particularly informative because per01 is effectively rescuing this phenotype in cryb. This can be understood in terms of the robust, high-amplitude oscillator in cryb, being less 'perturbable' by light as Cry photoreception is lost, whereas the damped oscillator in per01; cryb is more sensitive to the environmental stimulus, precisely because of its low amplitude. The damped oscillation in the per01; cryb double mutant can be eliminated by removing tim function, but this is temperature dependent, so tim cannot supply the full explanation for these residual cycles. Although these experiments have focused on the 'evening' oscillator, of related interest is that the residual 'morning' oscillator that anticipates the lights-on signal in per01 was also observed. It is clear that both of these studies raise again the possibility of an underlying rhythmicity in per01 flies that was initially suggested from statistical analyses of mutant locomotor records (Collins, 2005).

    The entrainment of a frequency-less oscillator in Neurospora crassa has been the subject of some recent debate, and the parallels with a residual rhythmicity in per-null Drosophila are striking. Furthermore, the rescue of per01 behavior by cryb would appear, at least superficially, to be similar to the situation in mammals in which a Cry mutation restores free-running rhythms to the arrhythmic mPer2 mutant mouse; this has been explained in terms of the freeing up in the double mutant of other mPer and Cry paralogues to interact and restore the original behavior. Since the fly does not have paralogues of per and cry, an explanation must be sought elsewhere. The only other genotypes identified so far with an anticipatory locomotor activity peak in LD and loss of rhythmicity in DD are disconnected (disco) and Pdf0. Neither mutation affects the molecular core of the circadian clock, rather the network of pacemaker neurons is disrupted. PDF is required for the functional integration of several clock neuronal groups within the brain, suggesting that disruption of interneuronal signaling causes arrhythmic behavioral output in the absence of synchronizing cues. In arrhythmic disco mutants, the clock gene expressing lateral neurons (LNvs and LNds) are usually absent, whereas the dorsal neurons are still present, thus indicating that the former are necessary for self-sustained rhythmicity, whereas the latter can only mediate rhythmic behavior under LD conditions (Collins, 2005).

    This networking of clock neurons provides a basis for possible models to explain LD behavioral anticipation in the absence of Per, based on functional differences between the three groups of clock genes expressing LNs. Of these, only the small ventral LNs (sLNvs) and dorsal LNs (LNds) have a self-sustaining molecular clock when initially released into DD, although the latter depends on the former for synchronization. The third group, the large ventral LNs (l-LNvs) do not have a self-sustaining clock, although after a few days, tim mRNA again begins to accumulate rhythmically in these cells. Furthermore, rhythmic Tim expression is more sensitive to disruption by cry mutations in the l-LNvs, than in the s-LNvs or the LNds under LD conditions, suggesting that rhythmic output from the l-LNvs are compromised in a cryb background. In turn, this may contribute to the peculiar defects of cryb that includes robust entrainment to LD cycles, but significantly reduces behavioral phase shifts to brief light pulses, and, unlike wild-type, the maintenance of rhythmic behavior in constant light (Collins, 2005).

    In the favored model, the robust s-LNv and LNd oscillators in cryb 'resist' the effects of brief light pulses, because of the impaired light input that is relayed to the s-LNvs, and from the s-LNvs to the LNds, by the more light-relevant l-LNvs. In per01, the molecular clock is severely dampened in all clock neurons, more so in the s-LNvs and LNds that have an endogenous cycle than the l-LNvs that do not. Thus, the light-mediated input from the l-LNv neurons into the s-LNvs, and indirectly to the LNds, is no longer resisted, and now overwhelms the residual damped per01 oscillator in these neurons, stimulating light-induced non-rhythmic locomotor behavioral output. However, when cryb and per01 are combined, the weak oscillator of per01 is no longer overcome by the light input because it is attenuated by cryb and mediated via the l-LNvs. Thus, rhythmic behavior is observed in LD cycles, providing a glimpse of the residual Per-independent, partly Tim-regulated clock. This model is preferred over a simpler one in which only the s-LNvs are involved, because previous studies have shown that the only direct photoreceptive input into these neurons is from the Hofbauer-Buchner eyelet, which is a very weak photoreceptor at best and it cannot, in the absence of other photoreceptors, entrain the fly's behavior (Helfrich-Forster, 2002). Thus, it is difficult to see how light information would be received by the s-LNvs to entrain the per01; cryb double mutant so effectively, unless it is transmitted from another neuronal source: the l-LNvs (Collins, 2005 and references therein).

    In support of the model, there appears to be both direct and indirect neural connections between the compound eyes and the l-LNvs, suggesting that the l-LNvs may act as the light 'amplifier'. This study extends earlier observations by showing that photoreceptor cells expressing the rhodopsin genes, Rh3 and Rh5, send their axons through the medulla terminating in close proximity to the general region where the l-LNvs likely extend their dendritic arborizations..Although not definitive, these results support earlier claims that the photoreceptors may directly (or indirectly) synapse with the l-LNvs. As stated above, these molecular and proposed functional differences between s- and l-LNvs may also contribute to explaining the loss of light responsiveness in cryb mutant flies, which are blind to constant light and brief light pulses, despite retaining light input from the canonical visual transduction pathway. Thus Cryptochrome, aside from being a photoreceptor in its own right, also appears to control a gateway for rhodopsin-mediated light input into the clock (Collins, 2005).

    Although the disruption of neural networks in this way probably explains the light responses of the clock in per01; cryb, it offers no molecular basis for the observed behavior. The loss of anticipation in tim-null-bearing genotypes suggests that Tim may play a key role. Although no significant nuclear Tim was observed in the LNvs or LNds of per01; cryb, the latter neurons being particularly relevant for providing the evening peak of locomotor activity present in the double mutants, it is suspected that Tim is shuttling continually in and out of the nucleus because Tim can enter the nucleus alone, but requires Per for nuclear retention, at least in larval clock neurons. Once in the nucleus, Tim is presumably interacting with as yet unidentified protein(s) in a light-dependent manner, generating behavioral rhythms in the double mutants. A microarray study found that 18 of the 72 genes that cycled in LD in wild-type also cycle in per01. Any one or more of these light-controlled proteins could therefore interact with Tim, contributing to the light-dependent oscillator of per01; cryb. In fact, it has been noted by others that a glutamine-rich transcriptional activator domain found within Tim may allow it to regulate other genes in a Per-independent manner (Collins, 2005).

    The period gene Thr-Gly polymorphism in Australian and African Drosophila melanogaster populations: implications for selection

    The period gene is a key regulator of biological rhythmicity in Drosophila. The central part of the gene encodes a dipeptide Thr-Gly repeat that has been implicated in the evolution of both circadian and ultradian rhythms. Length variation in the repeat follows a latitudinal cline in Europe and North Africa; this observation has now been extended to the southern hemisphere. A parallel cline is observed in Australia for one of the two major length variants and higher levels of some Thr-Gly length variants, particularly at the tropical latitudes, are found that are extremely rare in Europe. In addition >40 haplotypes from sub-Saharan Africa were examined and a very different and far more variable profile of Thr-Gly sequences was found. Statistical analysis of the periodicity and codon content of the repeat from all three continents reveals a possible mechanism that may explain how the repeat initially arose in the ancestors of the D. melanogaster subgroup of species. The results further reinforce the view that thermal selection may have contributed to shaping the continental patterns of Thr-Gly variability (Sawyer, 2006; full length of article).

    Nocturnal male sex drive in Drosophila

    Many behaviors and physiological processes including locomotor activity, feeding, sleep, mating, and migration are dependent on daily or seasonally reoccurring, external stimuli. In Drosophila, one of the best-studied circadian behaviors is locomotion. The fruit fly is considered a diurnal (day active/night inactive) insect, based on locomotor-activity recordings of single, socially naive flies 4 and 5. A new circadian paradigm has been developed that can simultaneously monitor two flies in simple social contexts. Heterosexual couples exhibit a drastically different locomotor-activity pattern than individual males, females, or homosexual couples. Specifically, female-male couples (FM) exhibit a brief rest phase around dusk but are highly active throughout the night and early morning. This distinct locomotor-activity rhythm is dependent on the clock genes and synchronized with close-proximity encounters, which reflect courtship, between the male and female. The close-proximity rhythm is dependent on the male and not the female and requires circadian oscillators in the brain and the antenna. Taken together, these data show that constant exposure to stimuli emanating from the female and received by the male olfactory and other sensory systems is responsible for the significant shift in intrinsic locomotor output of socially interacting flies (Fujii, 2007).

    Courtship behavior is mediated by the chemosensory, auditory, visual, and possibly mechanosensory systems. Because the close-proximity rhythm is maintained in DD, vision is likely to play no major role. To address the contribution of other senses, a series of experiments was performed in which the male was deprived of olfactory and auditory sensory modalities. Surgical removal of the aristae (main auditory organ) or the maxillary palps (minor olfactory organs) had no significant effect on close-proximity rhythm. However, when the third antennal segments were removed, or when males were homozygous mutants for the Or83b gene required for the detection of most volatile chemicals, frequency of close-proximity encounters was severely reduced at night and early morning. For both Or83b mutant flies and flies lacking the third antennal segment, the overall effects of time and genotype were significant by two-way ANOVA (p < 0.001), and the interaction between time and genotype was also significant for the former, but not the later. Taken together, these experiments indicate that olfaction is a major sensory modality required for an intense courtship rhythm (Fujii, 2007).

    Drosophila has multiple circadian clocks. The main clock is located in a small number of brain neurons and controls circadian locomotor activity in flies kept in social isolation. Peripheral clocks reside in numerous organs, including the antenna, eyes, and testes, and appear to control intrinsic circadian rhythms within each tissue context. For example, olfactory sensitivity exhibits a daily cycle, a phenomenon that is dependent on the appropriate cycling of the clock genes in olfactory neurons but independent of the cycling of these genes in the central brain neurons. To address the contributions of the central and peripheral clocks to close-proximity rhythms, the per 7.2:2 transgene, which confers per expression in parts of the brain sufficient for the maintenance of intrinsic locomotor activity of single males in isolation, was exploited. This transgene is not expressed in the antenna and therefore does not provide an oscillator for the olfactory sensory systems. FM couples in which the hemizygous per01 male did or did not contain the P7.2:2 transgene exhibited no close-proximity rhythm, indicating that per01 males' central clock, which mediates intrinsic locomotor rhythm in socially naive flies, is not sufficient for close-proximity rhythm. However, the possibility that loss of courtship rhythm is in part due to a slightly altered intrinsic rhythm in the central clock of these males cannot be completely excluded. It is also noted that a functional clock restricted to the antenna and sufficient for cycling olfactory sensitivity is not sufficient for close-proximity rhythm, despite the fact that cyc01 mutant males also show arrhythmic close-proximity behavior. Taken together, these findings are consistent with previous observations indicating that the mating rhythm in Drosophila is governed by clock genes (Fujii, 2007).

    A significant weakening was noted in the close-proximity rhythm in FM couples in which the male lacks olfactory input. One interpretation of these observations is that multiple sensory channels cooperate in establishing the close proximity rhythm but that the presence of at least one is sufficient for generating such a rhythm. Because a functional central pacemaker in the male is essential for the rhythm as well and a requirement for per and tim function in mating rhythms has been reported, it is proposed that external cues perceived by the olfactory and other sensory systems feed into the central pacemaker in the male to cause a shift in circadian activity. The ability of peripheral stimuli to reset the central pacemaker may be a male-specific feature because the shift is male-induced and independent of the female's phasing of the clock. It is interesting to speculate that sex-specific molecular and/or anatomical differences in the neural circuit of the central clock, also suggested by the different locomotor activities of single males and females, are responsible for the male-induced activity shift in a heterosexual social context (Fujii, 2007).

    The current studies show that rhythms in male sex drive and intrinsic male locomotor activity are out of phase, with highest male sex drive occurring throughout the subjective night and early morning when socially isolated flies rest. Interestingly, Drosophila olfactory responses oscillate in circadian fashion in the antenna independently of the central pacemaker neurons, with the highest sensitivity during the night. This study suggests that enhanced olfactory sensitivity might facilitate detection of predators, contribute to opportunistic feeding, or contribute to reproductive behaviors at times when flies are usually inactive; the third possibility is consistent with the increased, nocturnal male sex drive described in this study. Regardless of the biological significance, it seems likely that external chemical stimuli, which may also include allomones (a chemical substance, produced or acquired by an organism, that, when contacting an individual of another species in the natural context, evokes in the receiver a behavioral or physiological reaction adaptively favorable to the emitter) and food odors, as well as auditory and visual stimuli can fundamentally modify intrinsic locomotor activity (Fujii, 2007).

    How is the shift in locomotor activity brought about in the social context of heterosexual couples? One contributing factor may be that chemosensory detection and recognition of females that are encountered during the night (in the absence of major visual stimuli) is enhanced by a male olfactory system that is tuned to perform best during that period of the diurnal cycle. Interestingly, several male-specific genes expressed in the head have also been reported to be under circadian control, and at least one of them, sex-specific enzyme1, is expressed in the chemosensory system with peak activity during the night (Fujii, 2007).

    It is generally accepted that D. melanogaster is a diurnal insect. However, no studies investigating the circadian behavior of the fly in its natural environment have been reported. Regardless of whether nocturnal sex drive occurs under 'real-life' conditions, these findings establish that intrinsic locomotor activity is subject to extensive modification by external social cues. Ultimately, novel assays that consider the 'ecological realities' of Drosophila will have to be developed to reveal the largely unknown circadian behavior of this otherwise well-understood animal model system (Fujii, 2007).

    Circadian regulation in the ability of Drosophila to combat pathogenic infections

    This study sought to determine if the innate immune response is under circadian regulation and whether this impacts overall health status. To this end, infection of Drosophila with the human opportunistic pathogenic bacteria Pseudomonas aeruginosa was used as a model system. The survival rates of wild-type flies vary as a function of when, during the day, they are infected, peaking in the middle of the night. Although this rhythm is abolished in clock mutant flies, those with an inactive period gene are highly susceptible to infection, whereas mutants with impairment in other core clock genes exhibit enhanced survival. After an initial phase of strong suppression, the kinetics of bacterial growth correlate highly with time of day and clock mutant effects on survival. Expression profiling revealed that nighttime infection leads to a clock-regulated transient burst in the expression of a limited number of innate immunity genes. Circadian modulation of survival also was observed with another pathogen, Staphylococcus aureus. These findings suggest that medical intervention strategies incorporating chronobiological considerations could enhance the innate immune response, boosting the efficacy of combating pathogenic infections (Lee, 2008).

    Together, these findings suggest the following scenario for how the clock in Drosophila might influence the progression of an infection with P. aeruginosa. Early during the infection a robust immune response (perhaps both cellular and humoral) is mounted, which is effective in pathogen clearance irrespective of when during a daily cycle the flies are infected, as indicated by the rapid drop in bacterial titer during the first 5 hr postinfection. However, the clock regulates the induced levels of a limited subset of innate immunity players, such as PGRP-SA and drc, whereby infections in the middle of the night result in a transient burst early during the infection. Higher levels of a few key immune players over a certain threshold may contribute to keeping the titer of pathogenic bacteria low after the initial rapid-declining phase. By prolonging the suppression of bacterial growth during a critical window of the infection, this might provide an opportunity to mount or recruit additional host defenses in addition to AMPs, resulting in improved survival. Thus, the results suggest that the clock modulates the strength or responsiveness of immune activation in a time-of-day-dependent manner but only during a critical early phase of the infection process that has physiological consequences on the ability of the host to survive pathogenic infections. Indeed, it is noteworthy that P. aeruginosa eludes host defenses by the early suppression of antimicrobial peptide gene expression. It will be of interest to determine why the postinfection expression profiles of only certain immune response genes exhibit circadian regulation and how this is apparently restricted to a particular phase of the immune response (Lee, 2008).

    Although the time-of-day differences in the levels of induced peptidoglycan recognition protein PGRP-SA and drosocin (drc) are clearly consistent with the survival rates of wild-type flies infected at different times of day, this is not the case for the per01 and ClkJrk mutants in which the overall levels of drc are much lower in ClkJrk compared to per01 flies; a trend observed for other AMP genes surveyed. Although seemingly paradoxical, this is not unanticipated as there are precedents in the literature showing that flies can be more susceptible to bacterial infection despite elevated levels of AMP expression, indicating that excessive or inappropriate immune activation can be deleterious. In this context it is important to consider that besides the production of AMPs, innate immunity in adult Drosophila includes a proteolytic cascade leading to melanization and a cellular immune response characterized by phagocytosis. It is possible that inactivation of per might affect other host defense mechanisms that cannot be compensated by a potentially hypersensitive humoral immune response. Conversely, ClkJrk and other clock mutant flies might have a heightened activity of cellular immunity. Presently, the results, which are based on probing the expression profiles of several immune response genes, would seem to demand that the molecular mechanisms governing the time-of-day differences in survival for flies with functional clocks are different from those affecting survival rates in per01 and ClkJrk flies. Otherwise stated, it does not appear likely that the survival rates of per01 and ClkJrk flies are due simply to their clocks being pegged or held at a phase that is similar to either ZT/CT5 or ZT/CT17 in wild-type flies, respectively. Although future work will be required to resolve the molecular underpinnings governing the differential clock mutant effects on survival, these considerations suggest that core clock genes have 'non-circadian' related roles (i.e., roles not solely limited to their functions in timekeeping) in fighting microbial infections. Indeed, these findings add to a growing list of physiological and behavioral pathways that are differentially regulated in different clock mutants; e.g., mutations in per but not tim, Clk, or cyc play a key role in long-term memory formation in Drosophila (Lee, 2008).

    If the ability to evoke a stronger response at night enhances the efficacy of fighting a microbial infection, why restrict it to the night? It is widely thought that maintaining an optimal immune system is metabolically costly, competing for limited metabolic resources with other energetically demanding activities such as foraging or mating. Within this framework it is suggested that the clock might function as a temporal sieve to ensure the proper allocation of metabolic resources at biologically desirable times. From a more medical perspective, the results suggest that the innate immune system is a prime target for interventions based on chronobiological considerations in the hopes of boosting the ability to combat pathogenic infections (Lee, 2008).

    Circadian-clock-dependent rhythms in GPRK2 abundance control the rhythmic accumulation of ORs in OSN dendrites, which in turn control rhythms in olfactory responses

    The Drosophila circadian clock controls rhythms in the amplitude of odor-induced electrophysiological responses that peak during the middle of night. These rhythms are dependent on clocks in olfactory sensory neurons (OSNs), suggesting that odorant receptors (ORs) or OR-dependent processes are under clock control. Because responses to odors are initiated by heteromeric OR complexes that form odor-gated and cyclic-nucleotide-activated cation channels, whether regulators of ORs were under circadian-clock control was tested. The levels of G protein-coupled receptor kinase 2 (Gprk2) messenger RNA and protein cycle in a circadian-clock-dependent manner with a peak around the middle of the night in antennae. Gprk2 overexpression in OSNs from wild-type or cyc01 flies elicits constant high-amplitude electroantennogram (EAG) responses to ethyl acetate, whereas Gprk2 mutants produce constant low-amplitude EAG responses. ORs accumulate to high levels in the dendrites of OSNs around the middle of the night, and this dendritic localization of ORs is enhanced by GPRK2 overexpression at times when ORs are primarily localized in the cell body. These results support a model in which circadian-clock-dependent rhythms in GPRK2 abundance control the rhythmic accumulation of ORs in OSN dendrites, which in turn control rhythms in olfactory responses. The enhancement of OR function by GPRK2 contrasts with the traditional role of GPRKs in desensitizing activated receptors and suggests that GPRK2 functions through a fundamentally different mechanism to modulate OR activity (Tanoue, 2008).

    Many sensory systems are regulated by the circadian clock. Various insects including flies, moths, and cockroaches show circadian rhythms in odor-dependent electrophysiological and behavioral responses. In mammals, the firing rate of isolated mouse olfactory bulb neurons is regulated by the circadian clock, as are odor-evoked brain activity waves (e.g., event-related potentials [ERPs]) in humans. Daily rhythms in neuronal activity or sensitivity have been reported for other sensory systems, such as the visual and auditory systems (Tanoue, 2008).

    The circadian clock modulates olfactory responses in Drosophila: Robust electroantennogram (EAG) responses are seen during the middle of the night, and weak EAG responses are seen during the middle of the day (Krishnan, 1999). These rhythms in EAG responses are controlled by the olfactory sensory neurons (OSNs) in Drosophila, which act as independent peripheral circadian oscillators (Tanoue, 2004). Colocalization of the circadian oscillator and a rhythmic output to the OSNs indicates that the abundance and/or activity of odorant receptors (ORs) and/or OR-dependent processes are under clock control. Drosophila ORs are seven-transmembrane-domain proteins that share some structural similarities with G protein-coupled receptors (GPCRs). However, recent studies demonstrate that Drosophila ORs have an inverted membrane topology compared to canonical GPCRs and function as odor-gated and cyclic-nucleotide-activated cation channels. To understand how the clock modulates odor-dependent responses, it was determined whether factors that modulate ORs were regulated by the circadian clock (Tanoue, 2008).

    G protein-coupled receptor kinases (GPRKs) and arrestins act to terminate GPCR signaling in mammals, thereby protecting cells from receptor overstimulation. GPRK-phosophorylated GPCRs are internalized by arrestin and subsequently degraded or recycled. Two Gprk genes, Gprk1 and Gprk2, have been reported in Drosophila. Gprk1 messenger RNA (mRNA) is enriched in photoreceptor cells, and expression of a Gprk1 dominant-negative mutant in photoreceptors increases the amplitude of electroretinogram (ERG) responses (Lee, 2004). Gprk2 is required for egg and wing morphogenesis, as well as embryogenesis in Drosophila (Molnar, 2007; Schneider, 1997). In mammals, seven Gprk genes are divided into three subfamilies on the basis of sequence homology: the rhodopsin kinase or visual Gprk subfamily (Gprk1 and Gprk7), the β-adrenergic receptor kinase subfamily (Gprk2 and Gprk3), and the Gprk4 subfamily (Gprk4, Gprk5, and Gprk6). Gprk3 knockout mice are unable to mediate odor-induced desensitization of odorant receptors (Peppel, 1997). In contrast, loss of Gprk2 function in C. elegans olfactory sensory neurons results in reduced chemosensory behavior, suggesting that Gprk2 is necessary for GPCR signaling (Fukuto, 2004). These results suggest that GPRKs play different roles in vertebrate and invertebrate olfaction (Tanoue, 2008).

    This study reports that Gprk2 expression is regulated by circadian clocks in antennae and that GPRK2 drives circadian rhythms in olfactory responses by enhancing OR accumulation in the dendrites of basiconic sensilla. Gprk2 mRNA and protein expression levels were high around the middle of the night, coincident with the peak of olfactory responses. Flies that overexpress Gprk2 in OSNs show constant high EAG responses to ethyl acetate during 12 hr light:12 hr dark (LD) cycles and accumulate high levels of ORs in OSN dendrites, whereas hypomorphic Gprk2 mutants show constant low EAG responses to ethyl acetate during LD. On the basis of these results, it is proposed that GPRK2 mediates cycles of OR accumulation in OSN dendrites to generate rhythms in EAG responses (Tanoue, 2008).

    Gprk2 is a circadian output gene whose mRNA and protein peak during the middle of the night in antennae. This phase of mRNA expression is similar to that of per, tim, and other genes driven directly by CLK-CYC binding to E-box regulatory sequences. However, CLK-CYC-dependent genes are expressed at constitutively high levels in per01 and tim01 mutants and constitutively low levels in ClkJrk and cyc01 mutants, whereas Gprk2 is expressed at low levels in per01, tim01, and cyc01 mutants. Several rhythmically expressed transcripts identified by microarray analysis of heads have low levels of expression in per01 and ClkJrk mutants, but the mechanism governing their rhythmic expression has not been explored (Tanoue, 2008).

    Analysis of arrhythmic clock mutants indicates that cycling levels of Gprk2 mRNA give rise to rhythms in GPRK2 protein. The levels of GPRK2 protein cycle in phase with Gprk2 mRNA and correspond to rhythms in EAG responses. Other kinases such as Double-time (Dbt), Shaggy (Sgg)/GSK3, and Casein kinase 2 (CK2) in Drosophila are constitutively expressed proteins, whereas GPRK2 is the first example of a rhythmically expressed kinase. However, other kinases such as Erk-MAP kinase and Calcium/calmodulin-dependent protein kinase II in the chicken retina are rhythmically activated due to phosphorylation and control cGMP-gated ion channels in cone photoreceptors (Tanoue, 2008).

    Cycling levels of GPRK2 are coincident with rhythms in EAG responses; GPRK2 levels and EAG responses peak around the middle of the night and are at their lowest levels during the middle of the day. When GPRK2 levels are constitutively low, as in per01, tim01, and cyc01 mutants, EAG responses are also low. In addition, levels of GPRK2 are at or below the normal wild-type trough in Gprk26936 and Gprk2EY09213 mutants and generate EAG responses that are at or below those at the wild-type trough. GPRK2 levels are barely detectable in Gprk2pj1 antennae. These results suggest that Gprk2pj1 is not a null allele and raise the possibility that a Gprk2 null mutant will lack EAG responses altogether. Such a result would demonstrate that Gprk2 is required for olfactory responses per se. In contrast, constitutive overexpression of Gprk2 produces constant high EAG responses in both wild-type and cyc01 flies, demonstrating that high levels of GPRK2 can effect high amplitude EAG responses independent of other clock-dependent factors. Taken together, these results argue that Gprk2 levels control the amplitude of EAG responses. If so, this would imply that low levels of GPRK2 present in the Gprk26936 mutant do not cycle in abundance (Tanoue, 2008).

    Given that GPRK2 levels regulate the amplitude of EAG responses, what is the mechanism through which GPRK2 controls EAG response amplitude? The traditional targets of GPRKs are GPCRs. In the mammalian olfactory system, GPRK3 desensitizes ORs by triggering their internalization. These results suggest that Drosophila Gprk2 is necessary for EAG responses, and, taken together with C. elegans Gprk2 function, they indicate that GPRKs play a different role in invertebrate olfaction than in vertebrate olfaction. The subcellular localization of ORs is high in dendrites of basiconic sensilla at ZT17 and low at ZT5, but the abundance of ORs in these dendrites at ZT5 can be driven to high levels by increasing GPRK2 expression. These results support a model in which the circadian clock generates a rhythm of Gprk2 expression, which in turn generates rhythms in the amplitude of EAG responses by promoting OR accumulation (and consequently odor-gated cation-channel formation) in OSN dendrites from basiconic sensillae. GPRK2-dependent rhythms in the amplitude of spontaneous spikes are also seen in OSNs, thus demonstrating that the clock controls basic (i.e., odor-independent) properties of the OSN membrane. It is possible that the rhythmic localization of odor-gated cation channels to OSN dendrites accounts for rhythms in the amplitude of spontaneous spikes. The results can't exclude the possibility that cyclic expression of other genes also contribute to rhythms in EAG responses. mRNA cycling was tested for several genes that could potentially modulate EAG responses, including arrestin 2, Gprk1, and kurtz arrestin; arrestin 2 mRNA levels cycle, but neither Gprk1 or kurtz arrestin mRNA levels cycle. Given that microarray analysis was done on fly heads depleted of antennae, microarray analysis of antennae may reveal other rhythmically expressed genes that contribute to EAG rhythms (Tanoue, 2008).

    Myc-tagged ORs did not accumulate to high levels in the dendrites of trichoid sensilla at ZT17. Trichoid sensilla have different functions than basiconic sensilla; T1 trichoid sensilla detect the pheromone 11-cis-vaccenyl acetate (cVA), whereas the basiconic sensilla recognize food and plant odors. It could be that the circadian clock regulates OSN activity differently in basiconic sensilla and trichoid sensilla, although the possibility cannot be excluded that detection of Myc-tagged ORs in dendrites failed because of low expression levels in trichoid sensillae, poor permeability of Myc antibody into trichoid sensilla, or the long, thin geometry of trichoid sensillae (Tanoue, 2008).

    In summary, Drosophila Gprk2 mRNA and protein expression is under clock control in antennae. The levels of GPRK2 protein determine the amplitude of EAG responses to ethyl acetate in basiconic sensillae; high levels generate high-amplitude EAGs, and low levels produce low-amplitude EAGs. This result suggests that GPRK2 directly or indirectly enhances OR activity, in contrast to the inhibition of olfactory signaling by Gprk3 in mammals. Given that the most severe Drosophila Gprk2 mutant still produces low-amplitude EAG responses, a complete loss of Gprk2 function may lack EAG responses altogether and be required for olfaction. High levels of GPRK2 enhance OR localization to dendrites of basiconic sensillae and support a model in which rhythms in GPRK2 levels drive rhythms in OR localization to dendrites that ultimately mediates rhythms in EAG responses (Tanoue, 2008).

    Use-dependent plasticity in clock neurons regulates sleep need in Drosophila

    Sleep is important for memory consolidation and is responsive to waking experience. Clock circuitry is uniquely positioned to coordinate interactions between processes underlying memory and sleep need. Flies increase sleep both after exposure to an enriched social environment and after protocols that induce long-term memory. This study found that flies mutant for rutabaga, period, and blistered were deficient for experience-dependent increases in sleep. Rescue of each of these genes within the ventral lateral neurons (LNVs) restores increased sleep after social enrichment. Social experiences that induce increased sleep were associated with an increase in the number of synaptic terminals in the LNV projections into the medulla. The number of synaptic terminals was reduced during sleep and this decline was prevented by sleep deprivation (Donlea, 2009).

    Although sleep is a process that is necessary for survival, the functions of sleep are unknown. Sleep is regulated by circadian influences and is important for consolidation of long-term memory (LTM). Additionally, LTM is modulated by circadian mechanisms. Because the relationship between sleep, memory, and circadian rhythms seem to be phylogenetically conserved, Drosophila can be used to explain mechanisms that coordinate these processes. Drosophila show an increase in daytime sleep after exposure to socially enriched environments. Similarly, an increase in sleep after courtship conditioning is necessary for LTM (Donlea, 2009).

    Increased sleep after social enrichment is dependent upon genes that are required for learning and memory, including genes that alter cyclic adenosine monophosphate signaling. Although newly eclosed flies that are mutant for the adenylyl cyclase rutabaga (rut2080) show increased sleep after social enrichment, 3 to 4 day-old adult rut mutants do not respond to changes in the social environment. Elevating wild-type rut in adult flies with an RU486-inducible driver rescued experience-dependent increases in sleep in adult rut mutants; vehicle-treated siblings showed no increase in sleep. To identify circuits that mediate experience-dependent increases in sleep, a series of GAL4 lines was used to drive wild-type rut expression in brain circuits. Expression of UAS-rut using pdf-GAL4 restored the increase in daytime sleep and daytime sleep-bout duration, although to a lesser extent than GSelav. The expression pattern of pdf-GAL4 is limited to the ventral lateral neurons (LNVs), a group of clock neurons that express pigment-dispersing factor (pdf). Although pdf is the only known output from the LNVs, flies mutant for pdf show a wild-type increase in sleep (Donlea, 2009).

    Given this role of clock cells, the clock gene period (per), which is expressed in the LNVs and is required for LTM, was examined. Rescue of wild-type per using a 7.2-kb fragment of the per genomic sequence (per01; per+7.2-2) restored expression of PER at CT0 within the LNVs as well as the dorsal lateral neurons, LNDs; mutant flies carrying a null mutation, per01, expressed no PER. Although per01 mutants showed no increase in sleep after social enrichment, per01;per+7.2-2 flies displayed normal experience-dependent increases in sleep. per01 mutants have no LTM when tested 48 hours after training and only show a transient increase in sleep. per01;per+7.2-2 flies displayed LTM and increases in sleep. Although per levels are low in mutants for Clock and cycle, both acquire LTM and increase sleep after social enrichment. Thus, only a very small amount of per may be required to support increased sleep and LTM (Donlea, 2009).

    To further investigate the role of synaptic plasticity in clock cells, the Drosophila homolog for serum response factor (SRF), blistered (bs), was used. In mice, SRF is essential for activity-induced gene expression and plays an important role in synaptic long-term potentiation (Ramanan, 2005) and in contextual habituation (Etkin, 2006). bs retains a 93% identity with SRF within the DNA-binding MCM1-ARG80-Agamous-Deficiens-SRF (MADS) domain. Social enrichment elevated the transcription of bs in wild-type Canton-S (Cs) flies. Mutants carrying a P element inserted into the bs gene (P{GAL4}bs1348) do not increase sleep after social enrichment. This deficit was also found in flies carrying either of two other mutant alleles for bs (bs2 and bs3) and was present in flies that are homozygous for mutant bs alleles and flies that have been outcrossed to either Cs or to flies carrying the In(2LR)Px4 deficiency. The P-element insertion in bs1348 preserves the MADS domain; similar N-terminal truncated mutant SRF acts as dominant negative. BS is expressed throughout the brain, including pdf-expressing LNVs. When UAS-egfp was driven by P{GAL4}bs1348, expression was restricted to a small number of neurons, including the LNVs. Expression of bs using P{GAL4}bs1348 to drive either of two wild-type bs (UAS-bs) constructs rescued experience-dependent increases in sleep. Moreover, inducing bs expression within the LNVsusing pdf-GAL4 increased sleep after social enrichment (Donlea, 2009).

    To establish whether expression of bs is required for LTM, flies carrying the P{GAL4}bs1348 mutant allele were tested using courtship conditioning. Although P{GAL4}bs1348/+ flies acquire short-term memory, LTM was impaired. Rescue of wild-type bs using P{GAL4}bs1348 restored LTM. Next, the GAL4 repressor cry-GAL80 was used to block UAS-bs expression within the LNs. Although UAS-bs/+;cry-gal80/+ control flies showed significant courtship suppression, P{GAL4}bs1348/UAS-bs;cry-GAL80/+ flies had no LTM, which suggests a role for the LNs, although a role for the dorsal neurons (DNs) cannot be excluded. Although SRF deletion in mouse forebrain results in neurons with abnormal morphology (Knoll, 2006), the morphology of LNVs in mutant P{GAL4}bs1348/+ flies did not differ from that of LNVs in P{GAL4}bs1348/UAS-bs rescue flies All three mutants for bs had intact circadian rhythms and showed anticipatory activity before light-dark transitions; only bs3 flies show an altered period under constant darkness. These findings suggest that there are no developmental abnormalities in the LNVs in bs mutants (Donlea, 2009).

    Hypomorphic alleles for bs prevent proper wing development through interactions with Epidermal growth factor receptor (Egfr) signaling. Because Egfr alters sleep in Drosophila (Foltenyi, 2007), interactions between bs and Egfr may regulate responses to social experience. After social enrichment, transcription of Egfr was significantly elevated in Cs flies. The Egfr genomic sequence contains several CC(A/T)6GG CArG elements that can be bound by bs to promote transcription, and transcription of Egfr was significantly reduced in bs mutants. Thus, P{GAL4}bs1348 was used to drive expression of a constitutively active Egfr construct (UAS-Egfr*). Although P{GAL4}bs1348/+ mutants showed no change in sleep after social enrichment, activation of Egfr in P{GAL4}bs1348/+;UAS-Egfr*/+ flies increased sleep. Conversely, the expression of a dominant-negative construct for Egfr (UAS-EgfrDN) using pdf-GAL4 prevented increases in sleep after social enrichment (Donlea, 2009).

    A recent theory proposes that a function of sleep is to downscale synaptic connections. Moreover, structural plasticity can be induced by environmental manipulation in Drosophila. To quantify the effect of social enrichment on the number of post-synaptic terminals in LNV projections, pdf-GAL4 was used to drive expression of a green fluorescent protein (GFP)-tagged construct of the postsynaptic protein discs-large (UAS-dlgWT-gfp). After 5 days of social enrichment, LNV projections into the medulla of pdf-GAL4/+;;UAS-dlgWT-gfp/+ flies contained significantly more GFP-positive terminals. Although it has not been demonstrated that the labeled synaptic terminals are functional, these tools have been used to quantify synapses. The expression of the UAS-dlgWT-GFP marker did not alter synaptic function in a wild-type background and did not prevent the increase in sleep when expressed using pdf-GAL4 after social enrichment. To determine the effect of waking on synapse number, socially isolated pdf-GAL4/+;;UAS-dlgWT-gfp/+ flies and their enriched siblings either were allowed to sleep ad libitum or were sleep deprived for 48 hours after social enrichment. Although the number of dlg-GFP positive terminals remained elevated in sleep-deprived socially enriched flies, terminal number was significantly reduced in siblings that were allowed to sleep. Similarly, the number of presynaptic terminals in LNV projections into the medulla using a GFP-tagged construct of the presynaptic protein synaptobrevin (UAS-VAMP-GFP) in pdf-GAL4/+;UAS-VAMP-GFP/+ flies was increased. After 48 hours of recovery, socially enriched pdf-GAL4/+;UAS-VAMP-GFP/+ flies had a reduced number of VAMP-GFP-positive presynaptic terminals relative to their sleep-deprived siblings. A recent study has reported a clock-dependent remodeling in the axonal terminals of the PDF circuit that is highest during the day. Recent data indicates that hyperexcitation of a subset of the LNVs suppresses sleep in Drosophila. Together with the current results, these data suggest that the PDF circuit is well suited to test the hypothesis that sleep acts to downscale synaptic connections that are potentiated during waking experience (Donlea, 2009).

    Social experience modifies pheromone expression and mating behavior in male Drosophila melanogaster

    The social life of animals depends on communication between individuals. Recent studies in Drosophila demonstrate that various behaviors are influenced by social interactions. For example, courtship is a social interaction mediated by pheromonal signaling that occurs more frequently during certain times of the day than others. In adult flies, sex pheromones are synthesized in cells called oenocytes and displayed on the surface of the cuticle (see Cytology of adult Drosophila oenocytes). Although the role of Drosophila pheromones in sexual behavior is well established, little is known about the timing of these signals or how their regulation is influenced by the presence of other flies. This study reports that oenocytes contain functional circadian clocks that appear to regulate the synthesis of pheromones by controlling the transcription of desaturase1 (desat1), a gene required for production of male cuticular sex pheromones. Moreover, levels of these pheromones vary throughout the day in a pattern that depends on the clock genes and most likely also depends on the circadian control of desat1 in the oenocytes. To assess group dynamics, the genotypic composition of social groups (single versus mixed genotypes) were manipulated. This manipulation significantly affects clock gene transcription both in the head and oenocytes, and it also affects the pattern of pheromonal accumulation on the cuticle. Remarkably, it was found that flies in mixed social groups mate more frequently than do their counterparts in uniform groups. These results demonstrate that social context exerts a regulatory influence on the expression of chemical signals, while modulating sexual behavior in the fruit fly (Krupp, 2008).

    Social experience can influence behavior in Drosophila. In many cases, this influence is communicated via chemical cues, possibly in the form of pheromones. Individuals sense these social cues and respond to one another as they participate in group activities. Understanding how individuals send, receive, analyze, and respond to these signals is key to understanding how group dynamics affect behavior. By using measures of gene expression within the head and oenocytes, sex-pheromone accumulation on the outer cuticular surface, and mating behavior, this study has identified aspects of a mechanism driven by social experience that influences sexual behavior in Drosophila. This approach to studying group dynamics has reduced questions about social experience to questions about how molecular and cellular mechanisms mediate the effects of social interactions on individuals (Krupp, 2008).

    It was demonstrated that desat1 is a circadian output gene of a peripheral clock contained in the oenocytes. The presence of desat1 RNA and protein in the oenocytes supports the role of these cells as the primary site for the production of sex pheromones. Circadian fluctuations in the temporal display of many cuticular hydrocarbon compounds have been previously observed. This study has examined four of these compounds that act as sex pheromones. Consistent with the cyclic expression of desat1, the temporal profile of these pheromones are per dependent and appear to be influenced by a peripheral clock. Given the unique role oenocytes play in the production of sex pheromones, it is suggested that the temporal fluctuations in male sex-pheromone production are regulated by the oenocyte clock via the circadian regulation of desat1 (Krupp, 2008).

    Environmental inputs such as humidity and temperature are known to regulate hydrocarbons in Drosophila. This study shows that social interactions regulate hydrocarbon physiology. Such input from the physical and social environment may be mediated by hygro-, thermo-, and olfactory receptors in the antennae or by gustatory receptors in tarsi and proboscis. Whereas circadian clocks regulate olfactory and possibly gustatory input, as well as oenocyte output, it will be important to determine whether these clocks are required for the regulation of cuticular hydrocarbon as the fly adjusts to its environment in general (Krupp, 2008).

    In particular, why should sex-pheromone display be under circadian regulation? In Drosophila, sex pheromones differ between the sexes and are involved in mate recognition and preference. As such, these compounds represent sex-specific characters that may provide a fitness benefit when displayed at a particular concentration, in a particular blend, at specified times, and/or under certain conditions. All of these factors have been shown in various species to come into play when attempting to attract a mate. That sex-pheromone production and display are clock controlled implies that courtship and mating may have a temporal structure. Indeed, daily rhythmicity in courtship behavior was demonstrated by Hardeland some years ago. More recently, others have extended these studies and have shown a temporal pattern in both courtship and mating (Krupp, 2008).

    Sexually dimorphic and species-specific pheromones allow for mate recognition and effective mating strategies, and so may the circadian regulation of pheromones. In this way circadian changes in pheromonal profiles may represent the means of creating a temporal niche by influencing the probability of copulation In this regard, per has been associated with the temporal control of when different Drosophila species prefer to mate, thus creating a temporal barrier in mating. With this in mind, it will be important to determine whether the sex pheromones of other Drosophila species are also under circadian regulation, and whether the patterns differ from that of D. melanogaster. This might represent a mechanistic basis to the proposal, whereby populations exhibiting different pheromonal levels at different times might become reproductively isolated (Krupp, 2008).

    This study shows that social interactions influence the circadian regulation of male sex pheromones and the mating behavior. The overall levels of male sex pheromones as well as their temporal patterns are affected by social context. The mixture of two genotypes (WT and per0) within the group produced a general decrease in the total amount of monounsaturated hydrocarbon [except for 5-T and 7-tricosene or 7-C23:1 (7-T), which increase] and an increase in the frequency of mating. Given the prominent role male sex pheromones play in courtship behavior, it is possible that these changes in male pheromones directly relate to the change in mating behavior. 7-T is the most abundant of male sex pheromones in Drosophila and has been shown to increase female receptivity and repress male-male courtship. Males with elevated levels of 7-T show decreased latency to copulation and a higher mating success rate. Although an increase in 7-T may account for the increase in the frequency to copulate in these experiments, an alternative explanation is favored that a blend of hydrocarbons is associated with both the communication of social information and the influence on sexual behavior. It is inferred this because of the broad changes in cuticular hydrocarbons observed between different social contexts. It remains to be determined how a change in pheromone expression in response to social interactions affects mating behavior in the social assay (Krupp, 2008).

    Consistent with the circadian changes in sex-pheromone expression, social interactions also affected the molecular rhythm of the oenocyte clock and the expression of desat1. The amplitude of tim and Clk expression decreased, whereas that of per increased in response to the heterogeneous social grouping; the period and the phase of the molecular rhythms remained unchanged. Although the oenocyte clock remains rhythmic, the relationship between the expression levels of these core clock genes appeared altered. Transcriptional rhythms of the clock genes contribute to clock function, and changes in the amplitude of transcription have been shown to affect daily locomotor activity rhythms. Likewise, a change in the amplitude of clock gene expression within the oenocytes as a response to social interactions may drive the observed changes in sex-pheromone display. Correlated to the affect on Clk expression, the level of desat1 RNA was also reduced, thereby providing a putative mechanism whereby social experience can affect the circadian production of sex pheromones (Krupp, 2008).

    The patterns of clock gene expression within tissues of the head were also affected by social interactions. The expression level of per and Clk was decreased, tim remained unchanged, and again, the period and phase of clock gene expression was unaffected. The head contains multiple circadian oscillators, including the central clock cells, which are required for the generation of locomotor rhythms. Although whole-head preparations prevent localization of this effect to only the central clock, the changes in gene expression are intriguing in the context of previous findings, demonstrating an effect of social experience on locomotor activity rhythms. Together with the effect on the oenocytes, it would appear that social interactions affect multiple circadian systems, including both the central and peripheral clocks. Notably, these observations indicate that the amplitude of clock gene expression plays an important role in modulating both physiological (e.g., pheromone production) and behavioral (e.g., locomotor activity) rhythms (Krupp, 2008).

    A direct demonstration of the mechanistic links between the individual observations presented in this study (i.e., oenocyte clock > desat1 rhythm > pheromone rhythm > influences mating behavior) requires a means to manipulate the oenocyte clock specifically and the rhythmic expression desat1. This could be achieved through the use of the Gal4/UAS system targeting the oenocytes. However, it must be noted that the expression patterns of Gal4 driver lines used previously to examine the function of oenocytes are not restricted to only the oenocytes. In some cases, expression was observed in the brain and/or fat body, two tissues known to affect courtship and circadian behavior. Although there is no doubt about the use of these reagents to manipulate pheromones, it is concluded that the available oenocyte drivers are not adequate to discriminate oenocyte-specific effects on behavior or the temporal pattern of pheromone accumulation (Krupp, 2008).

    It is unlikely that social communication in Drosophila is limited to chemical signaling; indeed, tactile and auditory displays are important features of reproductive behavior, and it seems likely that a variety of sensory modalities are linked to pathways that mediate social responses. However, the data suggest that pheromonal responses are extremely sensitive to the social environment (Krupp, 2008).

    The possibility that behavioral feedback regulates molecular physiology in the fruit fly was proposed nearly two decades ago in the context of circadian rhythms. Interestingly, the same idea has been proposed in a quantitative theory that views the social environment as a selective pressure. This theory of indirect genetic effects relies on the idea that social interactions, occurring over generations, may direct the distribution of alleles within a population. According to this view, the relationship between an individual's phenotype and genotype is shaped in part by social context. This was observed in this study. Given the increase in mating associated with a mixed social grouping, the influence of social interactions on clock gene and desat1 expression may represent a detailed example in which an indirect genetic effect represents a modification in gene expression. The data may offer a glimpse into the effects of social interactions on the mechanisms of inheritance. The ability to quantify the effects of social and physical environmental influences on behavior, together with the powerful tools available for studying inherited mechanisms of behavior, suggests that Drosophila will be an important model organism for understanding the evolution of sociality (Krupp, 2008).

    Contribution of visual and circadian neural circuits to memory for prolonged mating induced by rivals

    Rival exposure causes Drosophila melanogaster males to prolong mating. Longer mating duration (LMD) may enhance reproductive success, but its underlying mechanism is currently unknown. This study found that LMD is context dependent and can be induced solely via visual stimuli. In addition, it was found that LMD involves neural circuits that are important for visual memory, including central neurons in the ellipsoid body, but not the mushroom bodies or the fan-shaped bodies, and may rely on the rival exposure memory lasting for several hours. LMD is affected by a subset of learning and memory mutants. LMD depends on the circadian clock genes timeless and period, but not Clock or cycle, and persists in many arrhythmic conditions. Moreover, LMD critically depends on a subset of pigment dispersing factor neurons rather than the entire circadian neural circuit. This study thus delineates parts of the molecular and cellular basis for LMD, a plastic social behavior elicited by visual cues (W. J. Kim, 2012).

    These findings provide evidence that males retain the memory of rival exposure, based primarily on visual stimuli, for several hours and lengthen mating duration accordingly. Indeed, LMD could be induced by allowing a male to view flies of either sex through a transparent partition, flies of different species or images of themselves in a mirror, indicating that LMD could be generated by visual stimuli without chemical communication. Not only could the LMD defects of per and tim mutants be rescued by the expression of PER or TIM via the nonrhythmic GAL4 driver, expression of PER in PDF neurons was sufficient to restore LMD to per mutants. Moreover, LMD requires electrical activity in lateral neurons, but not some of the dorsal neurons that are important for circadian rhythm. It therefore seems unlikely that circadian rhythm regulation is crucial for LMD. LMD involves the memory of rival exposure that lasts for several hours and is resistant to anesthesia and that it requires the rut function in the ellipsoid body. Finally, it was found that LMD generation depends on the activity of the compound eye, the PDF neurons and a subset of neurons in the ellipsoid body (W. J. Kim, 2012).

    Recent studies of social experience-mediated and context-dependent sexual behaviors of the fruit fly implicate chemical communication of males via pheromones as being important. This study found that vision in a social setting is also important for generating LMD. Although a recent report found that males use multiple redundant cues to detect mating rivals, this study found that LMD can be elicited by visual cues because rearing flies in constant darkness eliminated LMD, blind mutants and males with defective vision showed no LMD, and LMD can be generated simply by placing a mirror to allow a singly reared male fruit fly to see his reflection for 5 d. The visual stimulus for LMD likely derives from the red compound eye in motion because LMD can be induced by males of different species or females visible through a transparent film, but not by mutant males without red pigment in their compound eyes (W. J. Kim, 2012).

    LMD provides a new method for studying visual memory. To date, learning and memory studies in flies have focused primarily on the memory circuits in mushroom bodies; however, LMD requires a subset of neurons in the ellipsoid body rather than mushroom bodies. The ellipsoid body is the central brain region required for visual learning and memory, whereas mushroom bodies are not required for memory formation in visual learning in a flight simulator. Given that the mating duration assay is simpler than the flight simulator for the investigation of visual memory, it can be useful for large-scale genetic screens to identify mutants with altered visual memory (W. J. Kim, 2012).

    Cooperative Interaction between Phosphorylation Sites on PERIOD Maintains Circadian Period in Drosophila

    Circadian rhythms in Drosophila rely on cyclic regulation of the period (per) and timeless (tim) clock genes. The molecular cycle requires rhythmic phosphorylation of PER and TIM proteins, which is mediated by several kinases and phosphatases such as Protein Phosphatase-2A (PP2A) and the multi-isoformed Protein Phosphatase-1 (PP1), which was implicated in a photoperiod response in a previous study). This study used mass spectrometry to identify 35 'phospho-occupied' serine/threonine residues within PER, 24 of which are specifically regulated by PP1/PP2A. per transgenes were generated carrying phosphorylation site mutations and rescue of the per01 arrhythmic phenotype was tested. Surprisingly, most transgenes restore wild type rhythms despite carrying mutations in several phosphorylation sites. One particular transgene, in which T610 and S613 are mutated to alanine, restores daily rhythmicity, but dramatically lengthens the period to ∼30 hrs. Interestingly, the single S613A mutation extends the period by 2–3 hours, while the single T610A mutation has a minimal effect, suggesting these phospho-residues cooperate to control period length. Conservation of S613 from flies to humans suggests that it possesses a critical clock function, and mutational analysis of residues surrounding T610/S613 implicates the entire region in determining circadian period. A mutation at a previously identified site, S596, is largely epistatic to S613A, suggesting that S613 negatively regulates phosphorylation at S596. Together these data establish functional significance for a new domain of PER, demonstrate that cooperativity between phosphorylation sites maintains PER function, and support a model in which specific phosphorylated regions regulate others to control circadian period (Garbe, 2013)

    Sexual interactions influence the molecular oscillations in DN1 pacemaker neurons in Drosophila melanogaster

    Circadian rhythms can synchronize to environmental time cues, such as light, temperature, humidity, and food availability. Previous studies have suggested that these rhythms can also be entrained by social interactions. This study used Drosophila melanogaster as a model to study the influence of socio-sexual interactions on the circadian clock in behavior and pacemaker neurons. If two flies of opposite sex were paired and kept in a small space, the daily activity patterns of the two flies were clearly different from the sum of the activity of single male and female flies. Compared with single flies, paired flies were more active in the night and morning, were more active during females' active phase, and were less active during males' active phase. These behavioral phenotypes are related to courtship behavior, but not to the circadian clock. Nevertheless, in male-female pairs of flies with clocks at different speeds (wild-type and per S flies), clock protein cycling in the DN1 pacemaker neurons in the male brain were slightly influenced by their partners. These results suggest that sexual interactions between male-female couples can serve as a weak zeitgeber for the DN1 pacemaker neurons, but the effect is not sufficient to alter rhythms of behavioral activity (Hanafusa, 2013).

    Reported Drosophila courtship song rhythms are artifacts of data analysis

    In a series of landmark papers, Kyriacou, Hall, and colleagues reported that the average inter-pulse interval of Drosophila melanogaster male courtship song varies rhythmically (KH cycles), that the period gene controls this rhythm, and that evolution of the period gene determines species differences in the rhythm's frequency. Several groups failed to recover KH cycles, but this may have resulted from differences in recording chamber size. Using recording chambers of the same dimensions as used by Kyriacou and Hall, this study found no compelling evidence for KH cycles at any frequency. By replicating the data analysis procedures employed by Kyriacou and Hall, this study found that two factors (data binned into 10-second intervals and short recordings) imposed non-significant periodicity in the frequency range reported for KH cycles. Randomized data showed similar patterns. All of the results related to KH cycles are likely to be artifacts of binning data from short songs. Reported genotypic differences in KH cycles cannot be explained by this artifact and may have resulted from the use of small sample sizes and/or from the exclusion of samples that did not exhibit song rhythms (Stern, 2014).

    Role for circadian clock genes in seasonal timing: testing the bunning hypothesis

    A major question in chronobiology focuses around the 'Bunning hypothesis' which implicates the circadian clock in photoperiodic (day-length) measurement and is supported in some systems (e.g. plants) but disputed in others. This study used the seasonally-regulated thermotolerance of Drosophila melanogaster to test the role of various clock genes in day-length measurement. In Drosophila, freezing temperatures induce reversible chill coma, a narcosis-like state. Previous observations were have corroborated that wild-type flies developing under short photoperiods (winter-like) exhibit significantly shorter chill-coma recovery times (CCRt) than flies that were raised under long (summer-like) photoperiods. Arrhythmic mutant strains, per01, tim01 and ClkJrk, as well as variants that speed up or slow down the circadian period, disrupt the photoperiodic component of CCRt. The results support an underlying circadian function mediating seasonal daylength measurement and indicate that clock genes are tightly involved in photo- and thermo-periodic measurements (Pegoraro, 2014: PubMed).

    Relationships between the circadian system and Alzheimer's disease-Like symptoms in Drosophila

    Circadian clocks coordinate physiological, neurological, and behavioral functions into circa 24 hour rhythms, and the molecular mechanisms underlying circadian clock oscillations are conserved from Drosophila to humans. Clock oscillations and clock-controlled rhythms are known to dampen during aging; additionally, genetic or environmental clock disruption leads to accelerated aging and increased susceptibility to age-related pathologies. Neurodegenerative diseases, such as Alzheimer’s disease (AD), are associated with a decay of circadian rhythms, but it is not clear whether circadian disruption accelerates neuronal and motor decline associated with these diseases. To address this question, this study used transgenic Drosophila expressing various Amyloid-beta (Abeta; see Drosophila Appl) peptides, which are prone to form aggregates characteristic of AD pathology in humans. Development of AD-like symptoms were compared in adult flies expressing Abeta peptides in the wild type background and in flies with clocks disrupted via a null mutation in the clock gene period (per01). No significant differences were observed in longevity, climbing ability and brain neurodegeneration levels between control and clock-deficient flies, suggesting that loss of clock function does not exacerbate pathogenicity caused by human-derived Abeta peptides in flies. However, AD-like pathologies affected the circadian system in aging flies. Rest/activity rhythms were impaired in an age-dependent manner. Flies expressing the highly pathogenic arctic Abeta peptide showed a dramatic degradation of these rhythms in tune with their reduced longevity and impaired climbing ability. At the same time, the central pacemaker remained intact in these flies providing evidence that expression of Abeta peptides causes rhythm degradation downstream from the central clock mechanism (Long, 2014; PubMed).

    Associations between AD and impaired daily rhythms are well documented in humans, yet the causes and consequences of AD-related loss of circadian sleep/activity rhythms have not been teased apart. One of the unanswered questions is whether age-related decline of the circadian system contributes to AD progression. This study tested directly whether total arrhythmia caused by mutation in the core clock gene per would exacerbate AD-like phenotypes observed in an AD fly model. It was shown that premature death, progressive locomotor deficits, and vacuolization in the brain occurs with similar timing and intensity in flies with genetically disrupted clock mechanism as in control flies. Consistent with previous reports, the severity of symptoms is proportional to the pathogenicity of the expressed human Aβ fragments. However, within each genotype, symptoms in clock-deficient flies are similar to those in clock-competent flies. While this study's data show that disruption of the clock via removal of the core clock repressor PER does not exacerbate AD symptoms, it cannot be ruled out that disabling the positive clock arm could be more detrimental. A recent report showed that loss of the positive element BMAL1 causes brain neurodegeneration in mice. It was previously demonstrated that the loss of per accelerates death, locomotor impairments, and brain vacuolization in neurodegeneration-prone sniffer and swiss cheese fly mutants. However, the underlying molecular mechanism that mediates this effect is not known. The AD model used in this study is based on the expression of human Aβ peptides, which have been reported to accumulate into insoluble forms in aging flies. Because the disruption of the circadian clock does not affect the pathogenicity of these peptides, the study assumes that it has no effect on Aβ aggregation or clearance. In sum, this study's data show that the molecular and behavioral arrhythmia characteristic for per-null flies is not detrimental in this AD fly model (Long, 2014).

    However, the study shows that associations between AD and altered behavioral rhythms, observed in humans and AD model mice, also extend to fly AD models. Pan-neuronal expression of Aβ42 causes age-dependent impairment of circadian rest/activity rhythms, such that a reduced fraction of 50-days old elav>Aβ42 flies remain rhythmic in constant darkness compared to controls. A more dramatic disruption of circadian rhythms is observed in elav>Aβ42arc. In LD, 5-day old flies of this genotype show bimodal activity with an attenuated morning activity peak, while no activity peaks are detected in 15-day old flies, rather they are active around the clock, including nighttime when control flies have prolonged rest. In another related study, a loss of locomotor activity rhythms in elav>Aβ42arc flies even at young age was shown, similar to findings in this study. Together, these results demonstrate that AD model flies have rest/activity rhythm degradation reminiscent of the behavioral degradation observed in humans with AD (Long, 2014).

    Loss of rest/activity rhythms in elav>Aβ42arc flies formed the basis of investigation of the functional status of central pacemaker neurons, which are necessary and sufficient for the activity rhythms, at least in young flies. Immunocytochemistry of PDF-positive pacemaker neurons sLNv and lLNv shows the correct number and arborization pattern in elav>Aβ42arc flies. Moreover these neurons show nuclear peak and trough of the core clock protein PER indistinguishable from wild type flies. Similar observations have been published earlier, and it was additionally shown that even expression of the more pathogenic tandem Aβ42 construct leaves molecular oscillations in pacemaker neurons intact. Together, these data show dissociation between functioning molecular pacemaker and disrupted circadian coordination of rest/activity rhythms. This suggests that behavioral rhythm degradation observed in humans and mouse AD models may occur despite the presence of a functional central clock. Importantly, strong body temperature rhythms have been reported in AD patients again suggesting that the central clock may be intact in AD. This is reminiscent of the situation in very old flies and mammals, which show degradation of rest/activity rhythms while their central pacemaker neurons continue to show molecular oscillations (Long, 2014).

    While AD-related degradation of behavioral rhythms is not caused by malfunction of the central clock, other contributing factors remain to be investigated. Aβ related arrhythmicity might be due to non-cell-autonomous toxicity as focused expression of toxic peptides in clock containing cells does not affect behavioral rhythmicity, but expression outside of the pacemaker neurons may affect their synaptic connections. Additionally, downstream neuronal or humoral output pathways leading from the central pacemaker network to the motor centers could be adversely affected by Aβ aggregates. For example, recent studies reporting a direct measurement of neuronal activity in elav>Aβ42arc flies reveal increased latency and decreased response stability of the pathways leading from the giant fiber system in the brain into motor neurons of the thoracic ganglia. It is possible that neuronal deficits of this kind can disable output pathways from the central clock leading to fragmented rather than consolidated sleep. This may lead to a vicious cycle as sleep deprivation increases amyloid peptides in mice and Aβ aggregation disrupts the sleep/wake cycle. As flies provide a powerful toolkit to study both AD and circadian rhythms, studies at the intersection of chronobiology and AD should help to provide insights into the mechanisms underlying AD-related pathologies (Long, 2014).

    Dual PDF Signaling Pathways Reset Clocks Via TIMELESS and Acutely Excite Target Neurons to Control Circadian Behavior

    Molecular circadian clocks are interconnected via neural networks. In Drosophila, Pigment-Dispersing Factor (PDF) acts as a master network regulator with dual functions in synchronizing molecular oscillations between disparate PDF+ and PDF- circadian pacemaker neurons and controlling pacemaker neuron output. Yet the mechanisms by which PDF functions are not clear. This study has demonstrated that genetic inhibition of protein kinase A (PKA) in PDF- clock neurons can phenocopy PDF mutants, while activated PKA can partially rescue PDF receptor mutants. PKA subunit transcripts are also under clock control in non-PDF DN1p neurons. To address the core clock target of PDF, per was rescued in PDF neurons of arrhythmic per01 mutants. PDF neuron rescue induced high amplitude rhythms in the clock component Timeless (Tim) in per-less DN1p neurons. Complete loss of PDF or PKA inhibition also results in reduced Tim levels in non-PDF neurons of per01 flies. To address how PDF impacts pacemaker neuron output, PDF was focally applied to DN1p neurons and was found to acutely depolarize and increase firing rates of DN1p neurons. Surprisingly, these effects are reduced in the presence of an adenylate cyclase inhibitor, yet persist in the presence of PKA inhibition. Evidence is provided for a signaling mechanism (PKA) and a molecular target (Tim) by which PDF resets and synchronizes clocks; PDF exhibits an acute direct excitatory effect on target neurons to control neuronal output. The identification of Tim as a target of PDF signaling suggests it is a multimodal integrator of cell autonomous clock, environmental light, and neural network signaling. Moreover, these data reveal a bifurcation of PKA-dependent clock effects and PKA-independent output effects. Taken together, these results provide a molecular and cellular basis for the dual functions of PDF in clock resetting and pacemaker output (Seluzicki, 2014).

    Chronic jet lag impairs startle-induced locomotion in Drosophila
    PEndogenous circadian clocks with ~24-h periodicity are found in most organisms from cyanobacteria to humans. Daylight synchronizes these clocks to solar time. In humans, shift-work and jet lag perturb clock synchronization, and such perturbations, when repeated or chronic, are strongly suspected to be detrimental to healthspan. This study investigated locomotor aging and longevity in Drosophila melanogaster with genetically or environmentally disrupted clocks. Two mutations in period (per, a gene essential for circadian rhythmicity in Drosophila) were compared, after introducing them in a common reference genetic background: the arrhythmic per01, and perT which displays robust short 16-h rhythms. Compared to the wild type, both per mutants showed reduced longevity and decreased startle-induced locomotion in aging flies, while spontaneous locomotor activity was not impaired. The per01 phenotypes were generally less severe than those of perT, suggesting that chronic jet lag is more detrimental to aging than arrhythmicity in Drosophila. Interestingly, the adjustment of environmental light-dark cycles to the endogenous rhythms of the perT mutant fully suppressed the acceleration in the age-related decline of startle-induced locomotion, while it accelerated this decline in wild-type flies. Overall, these results show that chronic jet lag accelerates a specific form of locomotor aging in Drosophila, and that this effect can be alleviated by environmental changes that ameliorate circadian rhythm synchronization (Vaccaro, 2016).

    Do circadian genes and ambient temperature affect substrate-borne signalling during Drosophila courtship?

    Courtship vibratory signals can be air-borne or substrate-borne. They convey distinct and species-specific information from one individual to its prospective partner. This study focuses on the substrate-borne vibratory signals generated by the abdominal quivers of the Drosophila male during courtship; these vibrations travel through the ground towards courted females and coincide with female immobility. It is not known which physical parameters of the vibrations encode the information that is received by the females and induces them to pause. The intervals between each vibratory pulse were examined, a feature that was reported to carry information for animal communication. However, evidence of periodic variations in the lengths of these intervals could not be found, as has been reported for fly acoustical signals. Because it has been suggested that the genes involved in the circadian clock may also regulate shorter rhythms, effects of period on the interval lengths were determined. Males that were mutant for the period gene produce vibrations with significantly altered interpulse intervals; also, treating wild type males with constant light results in similar alterations to the interpulse intervals. These results suggest that both the clock and light/dark cycles have input into the interpulse intervals of these vibrations. By altering the interpulse intervals by other means, it was found that ambient temperature also has a strong effect. However, behavioural analysis suggests that only extreme ambient temperatures can affect the strong correlation between female immobility and substrate-borne vibrations (Medina, 2015).

    period-regulated feeding behavior and TOR signaling modulate survival of infection

    Most metazoans undergo dynamic, circadian-regulated changes in behavior and physiology. Currently, it is unknown how circadian-regulated behavior impacts immunity against infection. This study of behaviorally arrhythmic Drosophila circadian period mutants identifies a novel link between nutrient intake and tolerance of infection with B. cepacia, a bacterial pathogen of rising importance in hospital-acquired infections. Infection tolerance in wild-type animals was found to be stimulated by acute exposure to dietary glucose and amino acids. Glucose-stimulated tolerance was induced by feeding or direct injection; injections reveal a narrow window for glucose-stimulated tolerance. In contrast, amino acids stimulate tolerance only when ingested. The role of a known amino-acid-sensing pathway, the TOR (Target of Rapamycin) pathway, was investigated in immunity. TORC1 is circadian regulated and inhibition of TORC1 decreases resistance, as in vertebrates. Surprisingly, inhibition of the less well-characterized TOR complex 2 (TORC2) dramatically increases survival, through both resistance and tolerance mechanisms. This work suggests that dietary intake on the day of infection by B. cepacia can make a significant difference in long-term survival. TOR signaling mediates both resistance and tolerance of infection, and TORC2 was identified as a novel potential therapeutic target for increasing survival of infection (Allen, 2015).

    Codon usage affects the structure and function of the Drosophila circadian clock protein PERIOD

    Codon usage bias is a universal feature of all genomes, but its in vivo biological functions in animal systems are not clear. To investigate the in vivo role of codon usage in animals, this study took advantage of the sensitivity and robustness of the Drosophila circadian system. By codon-optimizing parts of Drosophila period (per), a core clock gene that encodes a critical component of the circadian oscillator, per codon usage was shown to be important for circadian clock function. Codon optimization of per results in conformational changes of the PER protein, altered PER phosphorylation profile and stability, and impaired PER function in the circadian negative feedback loop, which manifests into changes in molecular rhythmicity and abnormal circadian behavioral output. The study provides an in vivo example that demonstrates the role of codon usage in determining protein structure and function in an animal system. These results suggest a universal mechanism in eukaryotes that uses a codon usage "code" within genetic codons to regulate cotranslational protein folding (Fu, 2016).

    Reorganization of sleep by temperature in Drosophila requires light, the homeostat, and the circadian clock

    Increasing ambient temperature reorganizes the Drosophila sleep pattern in a way similar to the human response to heat, increasing daytime sleep while decreasing nighttime sleep. Mutation of core circadian genes blocks the immediate increase in daytime sleep, but not the heat-stimulated decrease in nighttime sleep, when animals are in a light:dark cycle. The ability of per01 flies to increase daytime sleep in light:dark can be rescued by expression of PER in either LNv or DN1p clock cells and does not require rescue of locomotor rhythms. Prolonged heat exposure engages the homeostat to maintain daytime sleep in the face of nighttime sleep loss. In constant darkness, all genotypes show an immediate decrease in sleep in response to temperature shift during the subjective day, implying that the absence of light input uncovers a clock-independent pro-arousal effect of increased temperature. Interestingly, the effects of temperature on nighttime sleep are blunted in constant darkness and in cryOUT mutants in light:dark, suggesting that they are dependent on the presence of light the previous day. In contrast, flies of all genotypes kept in constant light sleep more at all times of day in response to high temperature, indicating that the presence of light can invert the normal nighttime response to increased temperature. The effect of temperature on sleep thus reflects coordinated regulation by light, the homeostat, and components of the clock, allowing animals to reorganize sleep patterns in response to high temperature with rough preservation of the total amount of sleep (Parisky, 2016).

    Mutations in the circadian gene period alter behavioral and biochemical responses to ethanol in Drosophila

    Clock genes, such as period, which maintain an organism's circadian rhythm, can have profound effects on metabolic activity, including ethanol metabolism. In turn, ethanol exposure has been shown in Drosophila and mammals to cause disruptions of the circadian rhythm. Previous studies from our labs have shown that larval ethanol exposure disrupted the free-running period and period expression of Drosophila. In addition, a recent study has shown that arrhythmic flies show no tolerance to ethanol exposure. As such, Drosophila period mutants, which have either a shorter than wild-type free-running period (perS) or a longer one (perL), may also exhibit altered responses to ethanol due to their intrinsic circadian differences. This study tested the initial sensitivity and tolerance of ethanol exposure on Canton-S, perS, and perL, and then measured their Alcohol Dehydrogenase (ADH) and body ethanol levels. perL flies had slower sedation rate, longer recovery from ethanol sedation, and generated higher tolerance for sedation upon repeated ethanol exposure compared to Canton-S wild-type flies. Furthermore, perL flies had lower ADH activity and had a slower ethanol clearance compared to wild-type flies. The findings of this study suggest that period mutations influence ethanol induced behavior and ethanol metabolism in Drosophila and that flies with longer circadian periods are more sensitive to ethanol exposure (Liao, 2016).

    The clock gene period differentially regulates sleep and memory in Drosophila
    Circadian regulation is a conserved phenomenon across the animal kingdom, and its disruption can have severe behavioral and physiological consequences. Core circadian clock proteins are likewise well conserved from Drosophila to humans. While the molecular clock interactions that regulate circadian rhythms have been extensively described, additional roles for clock genes during complex behaviors are less understood. This study showed that mutations in the clock gene period (per) result in differential time-of-day effects on acquisition and long-term memory of aversive olfactory conditioning. Sleep is also altered in period mutants: while its overall levels don't correlate with memory, sleep plasticity in different genotypes correlates with immediate performance after training. This study further describes distinct anatomical bases for Period function by manipulating Period activity in restricted brain cells and testing the effects on specific aspects of memory and sleep. In the null mutant background, different features of sleep and memory are affected when a form of the period gene is reintroduce in glia, lateral neurons, and the fan-shaped body. The results indicate that the role of the clock gene period may be separable in specific aspects of sleep or memory; further studies into the molecular mechanisms of these processes suggest independent neural circuits and molecular cascades that mediate connections between the distinct phenomena (Fropf, 2018).

  • period: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity and Post-transcriptional Regulation | Protein Interactions | Developmental Biology | References

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