A new promoter element associated with daily time keeping in Drosophila.

Circadian clocks are autonomous daily timekeeping mechanisms that allow organisms to adapt to environmental rhythms as well as temporally organize biological functions. Clock-controlled timekeeping involves extensive regulation of rhythmic gene expression. To date, relatively few clock-associated promoter elements have been identified and characterized. In an unbiased search of core clock gene promoters from 12 species of Drosophila, we discovered a 29-bp consensus sequence that we designated as the Clock-Associated Transcriptional Activation Cassette or 'CATAC'. To experimentally address the spatiotemporal expression information associated with this element, we generated constructs with four separate native CATAC elements upstream of a basal promoter driving expression of either the yeast Gal4 or firefly luciferase reporter genes. Reporter assays showed that presence of wild-type, but not mutated CATAC elements, imparted increased expression levels as well as rhythmic regulation. Part of the CATAC consensus sequence resembles the E-box binding site for the core circadian transcription factor CLOCK/CYCLE (CLK/CYC), and CATAC-mediated expression rhythms are lost in the presence of null mutations in either cyc or the gene encoding the CLK/CYC inhibitor, period (per). Nevertheless, our results indicate that CATAC's enhancer function persists in the absence of CLK/CYC. Thus, CATAC represents a novel cis-regulatory element encoding clock-controlled regulation.

Temperature-dependent resetting of the molecular circadian oscillator in Drosophila.

Circadian clocks responsible for daily time keeping in a wide range of organisms synchronize to daily temperature cycles via pathways that remain poorly understood. To address this problem from the perspective of the molecular oscillator, we monitored temperature-dependent resetting of four of its core components in the fruitfly Drosophila melanogaster: the transcripts and proteins for the clock genes period (per) and timeless (tim). The molecular circadian cycle in adult heads exhibited parallel responses to temperature-mediated resetting at the levels of per transcript, tim transcript and TIM protein. Early phase adjustment specific to per transcript rhythms was explained by clock-independent temperature-driven transcription of per. The cold-induced expression of Drosophila per contrasts with the previously reported heat-induced regulation of mammalian Period 2. An altered and more readily re-entrainable temperature-synchronized circadian oscillator that featured temperature-driven per transcript rhythms and phase-shifted TIM and PER protein rhythms was found for flies of the 'Tim 4' genotype, which lacked daily tim transcript oscillations but maintained post-transcriptional temperature entrainment of tim expression. The accelerated molecular and behavioural temperature entrainment observed for Tim 4 flies indicates that clock-controlled tim expression constrains the rate of temperature cycle-mediated circadian resetting.

Adult circadian behavior in Drosophila requires developmental expression of cycle, but not period.

Circadian clocks have evolved as internal time keeping mechanisms that allow anticipation of daily environmental changes and organization of a daily program of physiological and behavioral rhythms. To better examine the mechanisms underlying circadian clocks in animals and to ask whether clock gene expression and function during development affected subsequent daily time keeping in the adult, we used the genetic tools available in Drosophila to conditionally manipulate the function of the CYCLE component of the positive regulator CLOCK/CYCLE (CLK/CYC) or its negative feedback inhibitor PERIOD (PER). Differential manipulation of clock function during development and in adulthood indicated that there is no developmental requirement for either a running clock mechanism or expression of per. However, conditional suppression of CLK/CYC activity either via per over-expression or cyc depletion during metamorphosis resulted in persistent arrhythmic behavior in the adult. Two distinct mechanisms were identified that may contribute to this developmental function of CLK/CYC and both involve the ventral lateral clock neurons (LN(v)s) that are crucial to circadian control of locomotor behavior: (1) selective depletion of cyc expression in the LN(v)s resulted in abnormal peptidergic small-LN(v) dorsal projections, and (2) PER expression rhythms in the adult LN(v)s appeared to be affected by developmental inhibition of CLK/CYC activity. Given the conservation of clock genes and circuits among animals, this study provides a rationale for investigating a possible similar developmental role of the homologous mammalian CLOCK/BMAL1 complex.

Recruitment of Cln3 cyclin to promoters controls cell cycle entry via histone deacetylase and other targets.

In yeast, the G1 cyclin Cln3 promotes cell cycle entry by activating the transcription factor SBF. In mammals, there is a parallel system for cell cycle entry in which cyclin dependent kinase (CDK) activates transcription factor E2F/Dp. Here we show that Cln3 regulates SBF by at least two different pathways, one involving the repressive protein Whi5, and the second involving Stb1. The Rpd3 histone deacetylase complex is also involved. Cln3 binds to SBF at the CLN2 promoter, and removes previously bound Whi5 and histone deacetylase. Adding extra copies of the SBF binding site to the cell delays Start, possibly by titrating Cln3. Since Rpd3 is the yeast ortholog of mammalian HDAC1, there is now a virtually complete analogy between the proteins regulating cell cycle entry in yeast (SBF, Cln3, Whi5 and Stb1, Rpd3) and mammals (E2F, Cyclin D, Rb, HDAC1). The cell may titrate Cln3 molecules against the number of SBF binding sites, and this could be the underlying basis of the size-control mechanism for Start.

Live Drosophila melanogaster Larvae Deter Oviposition by Drosophila suzukii.

The worldwide invasive insect pest, Drosophila suzukii Matsumura (spotted-wing Drosophila), lays eggs in soft and stone fruit before harvest. Hatched larvae cause fruit collapse and significant economic losses. Current control methods rely primarily on foliar insecticide applications, which are not sustainable long-term solutions due to regulatory restrictions and the risk of insecticide resistance developing. We showed before that D. suzukii were deterred from laying eggs on artificial media previously visited by its sister species-Drosophila melanogaster. In the current study, laboratory choice test experiments were conducted to identify which D. melanogaster life stage (eggs, larvae, or adult) deterred D. suzukii oviposition. We demonstrated that the presence of live D. melanogaster larvae on the egg-laying media consistently deterred D. suzukii oviposition. Drosophila melanogaster cuticular hydrocarbons (CHCs) were examined as candidate for the oviposition deterrent. CHCs of larval and adult D. melanogaster and D. suzukii were analyzed. In both species, the composition of the CHCs of larvae was similar to that of adults, although quantities present were much lower. Furthermore, the CHC profiles of the two species were markedly different. However, when assayed as deterrents in the laboratory choice test experiment, CHC extracts from D. melanogaster did not deter oviposition by D. suzukii.

Integration of light and temperature in the regulation of circadian gene expression in Drosophila.

Circadian clocks are aligned to the environment via synchronizing signals, or Zeitgebers, such as daily light and temperature cycles, food availability, and social behavior. In this study, we found that genome-wide expression profiles from temperature-entrained flies show a dramatic difference in the presence or absence of a thermocycle. Whereas transcript levels appear to be modified broadly by changes in temperature, there is a specific set of temperature-entrained circadian mRNA profiles that continue to oscillate in constant conditions. There are marked differences in the biological functions represented by temperature-driven or circadian regulation. The set of temperature-entrained circadian transcripts overlaps significantly with a previously defined set of transcripts oscillating in response to a photocycle. In follow-up studies, all thermocycle-entrained circadian transcript rhythms also responded to light/dark entrainment, whereas some photocycle-entrained rhythms did not respond to temperature entrainment. Transcripts encoding the clock components Period, Timeless, Clock, Vrille, PAR-domain protein 1, and Cryptochrome were all confirmed to be rhythmic after entrainment to a daily thermocycle, although the presence of a thermocycle resulted in an unexpected phase difference between period and timeless expression rhythms at the transcript but not the protein level. Generally, transcripts that exhibit circadian rhythms both in response to thermocycles and photocycles maintained the same mutual phase relationships after entrainment by temperature or light. Comparison of the collective temperature- and light-entrained circadian phases of these transcripts indicates that natural environmental light and temperature cycles cooperatively entrain the circadian clock. This interpretation is further supported by comparative analysis of the circadian phases observed for temperature-entrained and light-entrained circadian locomotor behavior. Taken together, these findings suggest that information from both light and temperature is integrated by the transcriptional clock mechanism in the adult fly head.

Selective entrainment of the Drosophila circadian clock to daily gradients in environmental temperature.

BACKGROUND: Circadian clocks are internal daily time keeping mechanisms that allow organisms to anticipate daily changes in their environment and to organize their behavior and physiology in a coherent schedule. Although circadian clocks use temperature compensation mechanisms to maintain the same pace over a range of temperatures, they are also capable of synchronizing to daily temperature cycles. This study identifies key properties of this process. RESULTS: Gradually ramping daily temperature cycles are shown here to synchronize behavioral and molecular daily rhythms in Drosophila with a remarkable efficiency. Entrainment to daily temperature gradients of amplitudes as low as 4 degrees C persisted even in the context of environmental profiles that also included continuous gradual increases or decreases in absolute temperature. To determine which elements of daily temperature gradients acted as the key determinants of circadian activity phase, comparative analyses of daily temperature gradients with different wave forms were performed. The phases of ascending and descending temperature acted together as key determinants of entrained circadian phase. In addition, circadian phase was found to be modulated by the relative temperature of release into free running conditions. Release at or close to the trough temperature of entrainment consistently resulted in phase advances. Re-entrainment to daily temperature gradients after large phase shifts occurred relatively slowly and required several cycles, allowing flies to selectively respond to periodic rather than anecdotal signals. The temperature-entrained phase relationship between clock gene expression rhythms and locomotor activity rhythms strongly resembled that previously observed for light entrainment. Moreover, daily temperature gradient and light/dark entrainment reinforced each other if the phases of ascending and descending temperature were in their natural alignment with the light and dark phases, respectively. CONCLUSION: The present study systematically examined the entrainment of clock-controlled behavior to daily environmental temperature gradients. As a result, a number of key properties of circadian temperature entrainment were identified. Collectively, these properties represent a circadian temperature entrainment mechanism that is optimized in its ability to detect the time-of-day information encoded in natural environmental temperature profiles. The molecular events synchronized to the daily phases of ascending and descending temperature are expected to play an important role in the mechanism of circadian entrainment to daily temperature cycles.

Control of daily transcript oscillations in Drosophila by light and the circadian clock.

The transcriptional circuits of circadian clocks control physiological and behavioral rhythms. Light may affect such overt rhythms in two ways: (1) by entraining the clock circuits and (2) via clock-independent molecular pathways. In this study we examine the relationship between autonomous transcript oscillations and light-driven transcript responses. Transcript profiles of wild-type and arrhythmic mutant Drosophila were recorded both in the presence of an environmental photocycle and in constant darkness. Systematic autonomous oscillations in the 12- to 48-h period range were detectable only in wild-type flies and occurred preferentially at the circadian period length. However, an extensive program of light-driven expression was confirmed in arrhythmic mutant flies. Many light-responsive transcripts are preferentially expressed in the compound eyes and the phospholipase C component of phototransduction, NORPA (no receptor potential), is required for their light-dependent regulation. Although there is evidence for the existence of multiple molecular clock circuits in cyanobacteria, protists, plants, and fungi, Drosophila appears to possess only one such system. The sustained photic expression responses identified here are partially coupled to the circadian clock and may reflect a mechanism for flies to modulate functions such as visual sensitivity and synaptic transmission in response to seasonal changes in photoperiod.

Control of Daily Locomotor Activity Patterns in Drosophila suzukii by the Circadian Clock, Light, Temperature and Social Interactions.

Understanding behavioral rhythms in a pest species can contribute to improving the efficacy of control methods targeting that pest. However, in some species, the behavioral patterns recorded in artificial conditions contrast greatly with observed wild-type behavioral rhythms. In this study, we identify the determinants of daily activity rhythms of the soft and stone fruit pest Drosophila suzukii. The impact of gender, space, social housing, temperature, light, fly morph, and the circadian clock on D. suzukii locomotor rhythms was investigated. Assays were performed under artificial laboratory conditions or more natural semifield conditions to identify how these factors affected daily locomotor behavior. Daily locomotor activity patterns collected under semifield conditions varied very little between the various sex and social condition combinations. However, in lab-based assays, individual and group-housed males often exhibited divergent activity patterns, with more prominent hyperactivity at light/dark transitions. In contrast, hyperactivity responses were suppressed under lab protocols mimicking summer conditions for groups of females and mixed-sex groups. Moreover, when environmental cues were removed, flies held in groups displayed stronger rhythmicity than individual flies. Thus, social interactions can reinforce circadian behavior and resist hyperactivity responses in D. suzukii. Fly morph appeared to have little impact on behavioral pattern, with winter and summer morph flies displaying similar activity profiles under April semifield and laboratory mimic environmental conditions. In conclusion, separate and combined effects of light, temperature, circadian clock function, and social interactions were apparent in the daily activity profiles of D. suzukii. When groups of female or mixed-sex flies were used, implementation of matching photoperiods and realistic daily temperature gradients in the lab was sufficient to re-create behavioral patterns observed in summer semifield settings. The ability to leverage lab assays to predict D. suzukii field behavior promises to be a valuable asset in improving control measures for this pest.

Potential of the European earwig (Forficula auricularia) as a biocontrol agent of the soft and stone fruit pest Drosophila suzukii.

BACKGROUND: The unintentional introduction of Drosophila suzukii (Matsumura) from Asia has caused global economic losses in the soft and stone fruit industries. Pesticide use can have unintended negative impacts on natural enemies, disrupting attempts to incorporate integrated pest management programmes. Generalist predators could potentially act as biocontrol agents of D. suzukii. In this context, the predatory capabilities of the European earwig (Forficula auricularia) were investigated. RESULTS: In semi-field conditions, F. auricularia were effective at reducing the reproductive rate of D. suzukii in more densely populated enclosures. In controlled laboratory conditions, significant negative effects of earwigs were observed for both low (three breeding pairs) and high (six breeding pairs) D. suzukii densities. Both semi-field and laboratory experiments revealed that F. auricularia predation on adult D. suzukii could not account for the subsequent reductions in population density. CONCLUSIONS: Reductions in both larval and adult offspring in the presence of earwigs indicate an impact on D. suzukii via predation prior to metamorphosis or disruption of oviposition. Although F. auricularia may predate D. suzukii populations, its capacity to act as a biocontrol agent may be limited. However, results suggest that F. auricularia may be a more effective biocontrol agent earlier in the growing season. © 2019 Society of Chemical Industry.

Recording and reproducing the diurnal oviposition rhythms of wild populations of the soft- and stone- fruit pest Drosophila suzukii.

Drosophila suzukii is a horticultural pest on a global scale which causes both yield and economic losses on a range of soft- and stone-fruit. Tackling this pest is problematic but exploiting behavioral rhythms could increase the impact of control. To do this, a better understanding of behavioral patterns is needed. Within this study we aimed to investigate rhythms in reproductive behavior of wild D. suzukii under natural conditions in the field. Environmental parameters were also recorded to decipher how they influence these rhythms. Assays were then performed on laboratory cultures, housed under artificial conditions mimicking the temperature and light cycles, to see if these patterns were reproducible and rhythmic. We were able to promote field like oviposition patterns within the laboratory using realistic temperature and light cycles regardless of variations in other factors including substrate, humidity, and lighting type. Locomotion activity was also recorded under these mimicked conditions to identify how this behavior interacts with oviposition rhythms. Both our field and laboratory assays show that oviposition behavior is likely under the control of the circadian clock and primarily occurs during the day. However, consistent with prior reports we observed that these patterns become crepuscular when day-time temperature peaks exceeded 30°C. This was also found within locomotion rhythms. With an increased understanding of how these behaviors are influenced by environmental conditions, we highlight the importance of using realistic temperature and light cycles when investigating behavioral patterns. From an increased understanding of D. suzukii behavior we increase our ability to target the pest in the field.

Reducing Drosophila suzukii emergence through inter-species competition.

BACKGROUND: Drosophila suzukii has dispersed widely from its native Asian range since 2008. Its arrival in the UK is resulting in economic losses in soft- and stone-fruit crops caused by larvae feeding on the flesh of ripening fruit. Although a large amount of research has been directed at controlling this pest, it is presently unknown how this invasive species interacts with native Drosophila species. RESULTS: In the work reported here, D. suzukii or Drosophila melanogaster adults were introduced to substrates pre-inoculated with the eggs of the same or the other species in a laboratory choice assay. Drosophila melanogaster adult emergence was not affected by pre-inoculation with D. suzukii. The rate of emergence of D. suzukii was significantly lower from medium pre-inoculated by D. melanogaster than from blank medium. In a subsequent experiment, significantly more D. suzukii eggs were laid in blank medium than in D. melanogaster pre-inoculated medium. CONCLUSION: The presence of D. melanogaster in a substrate significantly reduced D. suzukii emergence and egg laying. This study raises research questions about how this reduction mechanism is driven and how it could be exploited as part of future integrated pest management practices. © 2017 Society of Chemical Industry.

The G(1) cyclin Cln3 promotes cell cycle entry via the transcription factor Swi6.

In Saccharomyces cerevisiae (budding yeast), commitment to cell division in late G(1) is promoted by the G(1) cyclin Cln3 and its associated cyclin-dependent kinase, Cdc28. We show here that all known aspects of the function of Cln3 in G(1) phase, including control of cell size, pheromone sensitivity, cell cycle progress, and transcription, require the protein Swi6. Swi6 is a component of two related transcription factors, SBF and MBF, which are known to regulate many genes at the G(1)-S transition. The Cln3-Cdc28 complex somehow activates SBF and MBF, but there was no evidence for direct phosphorylation of SBF/MBF by Cln3-Cdc28 or for a stable complex between SBF/MBF and Cln3-Cdc28. The activation also does not depend on the ability of Cln3 to activate transcription when artificially recruited directly to a promoter. The amino terminus and the leucine zipper of Swi6 are important for the ability of Swi6 to respond to Cln3 but are not essential for the basal transcriptional activity of Swi6. Cln3-Cdc28 may activate SBF and MBF indirectly, perhaps by phosphorylating some intermediary protein.

Fluorescence/luminescence circadian imaging of complex tissues at single-cell resolution.

The use of luciferase reporter genes together with luminescence detection has enabled high frequency monitoring of molecular circadian clock function in living tissues. With the help of an intensified CCD camera combined with an inverted epifluorescence microscope, the authors have established a new imaging strategy that makes use of transgenic cell type-specific expression of fluorescent proteins to identify cells of interest for subsequent circadian luminescence recording at single-cell resolution.

The right period for a Siesta.

Drosophila melanogaster has a broad geographic range. Daily activity in this species exhibits seasonality such that midday rest expands on long warm days, possibly to avoid desiccation. Comparative analyses show that temperature-dependent control of this behavior is partly linked to patterns of per mRNA splicing that are absent in Drosophila yakuba, a related species native to warmer climates with little seasonal change.

Interplay of circadian clocks and metabolic rhythms.

This review examines the connections between circadian and metabolic rhythms. Examples from a wide variety of well-studied organisms are used to illustrate some of the genetic and molecular pathways linking circadian timekeeping to metabolism. The principles underlying biological timekeeping by intrinsic circadian clocks are discussed briefly. Genetic and molecular studies have unambiguously identified the importance of gene expression feedback circuits to the generation of overt circadian rhythms. This is illustrated particularly well by the results of genome-wide expression studies, which have uncovered hundreds of clock-controlled genes in cyanobacteria, fungi, plants, and animals. The potential connections between circadian oscillations in gene expression and circadian oscillations in metabolic activity are a major focus of this review.

Molecular and statistical tools for circadian transcript profiling.

This article describes methods used to evaluate mRNA expression patterns on microarrays and their application in circadian biology. With the intention of complementing rather than duplicating the existing literature, particular emphasis is placed on experimental design, data analysis techniques, and independent verification. Both comparative and temporal study designs are discussed, and their use in circadian research is illustrated with examples. Data analysis methods to assess periodic components in time series data are outlined in detail.

Molecular genetics of timing in intrinsic circadian rhythm sleep disorders.

Recent advances in circadian biology are identifying key genes and the molecular clockworks they command. These biochemical systems provide new tools for evaluating clinically observed, intrinsic circadian rhythm sleep disorders. A striking example was last year's discovery of a point mutation in a human clock gene that produces a sleep phase syndrome. This finding suggested that other intrinsic sleep disorders may have genetic underpinnings, and that less debilitating variations in sleep/wake behavior may be revealed by molecular screening of known clock genes in broader human populations.