A neural network underlying circadian entrainment and photoperiodic adjustment of sleep and activity in Drosophila

A sensitivity of the circadian clock to light/dark cycles ensures that biological rhythms maintain optimal phase relationships with the external day. In animals, the circadian clock neuron network (CCNN) driving sleep/activity rhythms receives light input from multiple photoreceptors, but how these photoreceptors modulate CCNN components is not well understood. Here we show that the Hofbauer-Buchner eyelets differentially modulate two classes of ventral lateral neurons (LNvs) within the Drosophila CCNN. The eyelets antagonize Cryptochrome (CRY)- and compound-eye-based photoreception in the large LNvs while synergizing CRY-mediated photoreception in the small LNvs. Furthermore, we show that the large LNvs interact with subsets of “evening cells” to adjust the timing of the evening peak of activity in a day length-dependent manner. Our work identifies a peptidergic connection between the large LNvs and a group of evening cells that is critical for the seasonal adjustment of circadian rhythms. SIGNIFICANCE STATEMENT In animals, circadian clocks have evolved to orchestrate the timing of behavior and metabolism. Consistent timing requires the entrainment these clocks to the solar day, a process that is critical for an organism's health. Light cycles are the most important external cue for the entrainment of circadian clocks, and the circadian system uses multiple photoreceptors to link timekeeping to the light/dark cycle. How light information from these photorecptors is integrated into the circadian clock neuron network to support entrainment is not understood. Our results establish that input from the HB eyelets differentially impacts the physiology of neuronal subgroups. This input pathway, together with input from the compound eyes, precisely times the activity of flies under long summer days. Our results provide a mechanistic model of light transduction and integration into the circadian system, identifying new and unexpected network motifs within the circadian clock neuron network

Unique features of a global human ectoparasite identified through sequencing of the bed bug genome

The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host–symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human–bed bug and symbiont–bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite

The circadian clock of Drosophila going wild: PERIOD and TIMELESS oscillations under natural conditions

The circadian clock of the fruit fly Drosophila melanogaster relies on 7 groups of clock neurons per brain hemisphere which are bilaterally clustered in dorsal (DN1s, DN2s and DN3s) and lateral (s-LNvs, l-LNvs, LNds and LPNs) according to their positions in the brain. In these neurons, clock genes such as period (per) and timeless (tim) operate in interlocked feed-back loops. Under rectangular 12:12 light:dark (LD) regimes and constant temperature PER and TIM proteins start to accumulate in the cytoplasm of all clock neurons in the middle of the night and reach their maximum levels at the end of the dark phase. At lights-on TIM is degraded in a light dependent manner; in the absence of TIM, PER is also degraded. To date, almost all behavioural and molecular analyses of fly circadian rhythmicity have been carried out in the laboratory. Nevertheless laboratory conditions do not reflect the complexity of the stimuli that are present in the natural environment. In 2006 our lab started a research project (granted by the European Commission, 6th Framework Programme; Project EUCLOCK N° 018741), in collaboration with the group of Prof. C. P: Kyriacou at the Department of Genetics, University of Leicester (UK), dealing with the characterization of the circadian clock of D. melanogaster under real natural conditions. To investigate the functioning of the circadian clock in a real natural environment we studied PER and TIM expression in the circadian clock neurons of flies exposed to natural conditions throughout 2008 and 2009. The flies analysed belong to the WT-ALA (Wild Type ALto Adige) strain of D. melanogaster which has been established in 2006 from a natural population sampled in the North of Italy. Upon analysis of PER and TIM expression profiles within the clock neurons of fruit flies experiencing real natural environment we found that, unexpectedly, PER and TIM oscillations appear to be decoupled in certain circumstances. In fact, under long and hot days as in Summer conditions (Natural LD~ 15:9; Tmax~35°C; Tmin~25°C) the peak of PER is advanced and the peak of TIM delayed, leading to an oscillation almost in antiphase. Moreover, we hypothesize that the decoupling in PER and TIM oscillation profiles is linked to the decoupling in the phase of the morning and evening burst of activity also observed under natural conditions (Bhutani et al., submitted). In addition, we observed that, irrespectively of the season, the peak of PER within the DN1s is always advanced compared to that within the lateral cells and this phase advance do not depends on PDF signaling. Once the molecular oscillation profiles of PER and TIM under natural conditions were revealed, we attempted to reproduce our findings under laboratory conditions. This part of the project was achieved thanks to the collaboration with Prof. Charlotte Helfrich-Förster's laboratory, at the Department of Neurobiology and Genetics, University of Würzburg (DE). In particular, wild type flies were entrained to laboratory LD 16:8 regimes in which the slow increasing and decreasing in light intensity typical of natural dawns and dusks was simulated, at two different constant temperatures (20°C and 30°C). We confirmed that the huge phase shift between PER and TIM oscillations which characterizes natural Summer days is caused, at least in part, by high temperatures, albeit natural thermocycles appear to be stronger environmental cues than constant high temperatures. Moreover, we observed that the phase advance in PER cycling within the DN1s hold true in the lab under the particular conditions we used for the entrainment. Therefore, we hypothesize that this phase advance is mainly a response to a specific environmental conditions, namely the ramping in light intensities we used to simulate sunrise and sunset.La sede anatomica del core dell’orologio circadiano di D. melanogaster è costituita da circa 100 neuroni per emisfero cerebrale, suddivisi in 7 gruppi: 3 gruppi di neuroni dorsali (DN1s, DN2s e DN3s) e 4 gruppi di neuroni laterali (s-LNvs, l-LNvs, LNds, e LPNs). A livello molecolare, l'orologio circadiano consta di un sistema di loop a retroazione negativa interconnessi tra loro. In condizioni standard di laboratorio, ovvero in regimi di LD 12:12 e a temperatura costante, i geni period (per) e timeless (tim) vengono trascritti ad opera dei fattori di trascrizione CLOCK (CLK) e CYCLE (CYC). Le proteine PERIOD (PER) e TIMELESS (TIM) si accumulano durante la notte e raggiungono un picco alla fine della notte/inizio del giorno, in maniera sincrona in tutti i neuroni orologio. Verso la fine della notte, PER e TIM entrano nel nucleo, dove inibiscono la trascrizione degli stessi geni che li codificano (per e tim). All'accensione della luce i livelli di TIM scendono rapidamente a causa della degradazione luce dipendente mediata dal fotorecettore per la luce blu CRYPTOCHROME (CRY). In assenza di TIM, anche PER va incontro a degradazione. Ad oggi, gli studi sull'orologio circadiano di Drosophila sono stati condotti esclusivamente in condizioni di laboratorio, anche se in qualche caso si è cercato di riprodurre le caratteristiche dell'ambiente naturale. Tuttavia, gli stimoli ambientali a cui i moscerini sono esposti in laboratorio sono di gran lunga meno complessi degli stimoli realmente presenti in natura. Nell'ambiente naturale la luce cambia continuamente, sia per quanto riguarda la sua intensità che la composizione del suo spettro ed anche la temperatura è soggetta a variazioni continue più o meno accentuate a seconda della stagione e della latitudine. Nel 2006 il nostro laboratorio ha avviato un progetto di ricerca (EU Project EUCLOCK N° 018741, 6th Framework Programme) in collaborazione con il gruppo del Prof. C.P. Kyriacou (Department of Genetics, University of Leicester, UK) con l'obiettivo di studiare e caratterizzare il funzionamento dell'orologio circadiano di Drosophila in condizioni naturali. In questo lavoro sono riportati i risultati della sperimentazione condotta nell'ambito della mia tesi di dottorato che si è concentrato sull'analisi dei profili di oscillazione delle proteine PER e TIM nei diversi neuroni orologio di moscerini esposti a condizioni naturali. Il ceppo di moscerini selvatici utilizzato negli esperimenti (WT-ALA) è stato stabilizzato a partire da una collezione di linee isofemminili campionate nel 2006 nel Nord Italia (Val Venosta, BZ). L'analisi dei dati ottenuti nelle diverse condizioni ambientali analizzate, (rappresentative delle quattro stagioni) ci ha permesso di rilevare che i profili di oscillazione di PER e TIM appaiono disaccoppiati in certe condizioni ambientali. Infatti, abbiamo riscontrato che quando le giornate sono lunghe e calde (Primavera/Estate) il picco di PER è ritardato rispetto a quello che si osserva in giornate più corte e fresche (Autunno) mentre quello di TIM sembra anticipare. Questi spostamenti dei picchi delle due proteine fanno si che, in particolare durante l'Estate, PER e TIM mostrino il loro massimo di espressione in alcuni gruppi di neuroni orologio con quasi 12 ore di differenza; PER e TIM sembrano quindi oscillare in antifase. Abbiamo osservato inoltre che, indipendentemente dalle condizioni ambientali cui sono esposti i moscerini, PER raggiunge il picco di espressione prima nei DN1s e successivamente nei neuroni orologio laterali. Abbiamo inoltre dimostrato che questa anticipazione dei DN1s è indipendente dal neuropeptide PDF, prodotto da s-LNvs e l-LNvs e implicato nell'output dell'orologio circadiano. Dopo aver descritto in dettaglio i profili di oscillazione di PER e TIM in condizioni naturali, abbiamo ritenuto opportuno, al fine di identificare le componenti ambientali responsabili dei fenomeni osservati, cercare di riprodurre in laboratorio i risultati ottenuti, apportando alcune modificazioni mirate ai tradizionali profili (rettangolari) di LD e alle temperature utilizzati negli esperimenti di laboratorio. Questi esperimenti sono stati condotti in collaborazione con il gruppo della Prof.ssa C. Helfrich-Förster (Department of Neurobiology and Genetics, University of Würzburg, DE). Abbiamo esposto i moscerini a cicli di LD 16:8 e simulato albe e tramonti mediante variazioni controllate dell'intensità luminosa, a due differenti temperature costanti (20°C e 30°C). Con questi esperimenti abbiamo potuto confermare quanto avevamo ipotizzato analizzando i risultati ottenuti in condizioni naturali, ovvero che lo spostamento delle fasi dei picchi di PER e TIM sia non solo determinato dal fotoperiodo ma, almeno in parte, anche dalla temperatura. Inoltre, tali esperimenti ci hanno permesso di collegare l'anticipazione del picco di PER nei DN1s al graduale aumento o diminuzione dell'intensità luminosa che si verifica normalmente durante l'alba e il tramonto

Data from: Normal vision can compensate for the loss of the circadian clock.

Circadian clocks are thought to be essential for timing the daily activity of animals, and consequently increase fitness. This view was recently challenged for clock-less fruit flies and mice that exhibited astonishingly normal activity rhythms under outdoor conditions. Compensatory mechanisms appear to enable even clock mutants to live a normal life in nature. Here, we show that gradual daily increases/decreases of light in the laboratory suffice to provoke normally timed sharp morning (M) and evening (E) activity peaks in clock-less flies. We also show that the compound eyes, but not Cryptochrome (CRY), mediate the precise timing of M and E peaks under natural-like conditions, as CRY-less flies do and eyeless flies do not show these sharp peaks independently of a functional clock. Nevertheless, the circadian clock appears critical for anticipating dusk, as well as for inhibiting sharp activity peaks during midnight. Clock-less flies only increase E activity after dusk and not before the beginning of dusk, and respond strongly to twilight exposure in the middle of the night. Furthermore, the circadian clock responds to natural-like light cycles, by slightly broadening Timeless (TIM) abundance in the clock neurons, and this effect is mediated by CRY

Raw Data Schlichting et al

Raw Data Schlichting et a

Drosophila RSK Influences the Pace of the Circadian Clock by Negative Regulation of Protein Kinase Shaggy Activity

Endogenous molecular circadian clocks drive daily rhythmic changes at the cellular, physiological, and behavioral level for adaptation to and anticipation of environmental signals. The core molecular system consists of autoregulatory feedback loops, where clock proteins inhibit their own transcription. A complex and not fully understood interplay of regulatory proteins influences activity, localization and stability of clock proteins to set the pace of the clock. This study focuses on the molecular function of Ribosomal S6 Kinase (RSK) in the Drosophila melanogaster circadian clock. Mutations in the human rsk2 gene cause Coffin–Lowry syndrome, which is associated with severe mental disabilities. Knock-out studies with Drosophila ortholog rsk uncovered functions in synaptic processes, axonal transport and adult behavior including associative learning and circadian activity. However, the molecular targets of RSK remain elusive. Our experiments provide evidence that RSK acts in the key pace maker neurons as a negative regulator of Shaggy (SGG) kinase activity, which in turn determines timely nuclear entry of the clock proteins Period and Timeless to close the negative feedback loop. Phosphorylation of serine 9 in SGG is mediated by the C-terminal kinase domain of RSK, which is in agreement with previous genetic studies of RSK in the circadian clock but argues against the prevailing view that only the N-terminal kinase domain of RSK proteins carries the effector function. Our data provide a mechanistic explanation how RSK influences the molecular clock and imply SGG S9 phosphorylation by RSK and other kinases as a convergence point for diverse cellular and external stimuli

Pigment-Dispersing Factor-expressing neurons convey circadian information in the honey bee brain

Pigment-Dispersing Factor (PDF) is an important neuropeptide in the brain circadian network of Drosophila and other insects, but its role in bees in which the circadian clock influences complex behaviour is not well understood. We combined high-resolution neuroanatomical characterizations, quantification of PDF levels over the day and brain injections of synthetic PDF peptide to study the role of PDF in the honey bee Apis mellifera. We show that PDF co-localizes with the clock protein Period (PER) in a cluster of laterally located neurons and that the widespread arborizations of these PER/PDF neurons are in close vicinity to other PER-positive cells (neurons and glia). PDF-immunostaining intensity oscillates in a diurnal and circadian manner with possible influences for age or worker task on synchrony of oscillations in different brain areas. Finally, PDF injection into the area between optic lobes and the central brain at the end of the subjective day produced a consistent trend of phase-delayed circadian rhythms in locomotor activity. Altogether, these results are consistent with the hypothesis that PDF is a neuromodulator that conveys circadian information from pacemaker cells to brain centres involved in diverse functions including locomotion, time memory and sun-compass orientation