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Animals possess a circadian central clock in the brain, where circadian behavioural rhythms are generated. In the fruit fly (Drosophila melanogaster), the central clock comprises a network of approximately 150 clock neurons, which is important for the maintenance of a coherent and robust rhythm. Several neuropeptides involved in the network have been identified, including Pigment-dispersing factor (PDF) and CCHamide1 (CCHa1) neuropeptides. PDF signals bidirectionally to CCHa1-positive clock neurons; thus, the clock neuron groups expressing PDF and CCHa1 interact reciprocally. However, the role of these interactions in molecular and behavioural rhythms remains elusive. In this study, we generated Pdf (01) and CCHa1(SK8) double mutants and examined their locomotor activity-related rhythms. The single mutants of Pdf (01) or CCHa1(SK8) displayed free-running rhythms under constant dark conditions, whereas approximately 98% of the double mutants were arrhythmic. In light-dark conditions, the evening activity of the double mutants was phase-advanced compared with that of the single mutants. In contrast, both the single and double mutants had diminished morning activity. These results suggest that the effects of the double mutation varied in behavioural parameters. The double and triple mutants of per (01), Pdf (01), and CCHa1(SK8) further revealed that PDF signalling plays a role in the suppression of activity during the daytime under a clock-less background. Our results provide insights into the interactions between PDF and CCHa1 signalling and their roles in activity rhythms.

Animals possess a circadian central clock in the brain, where circadian behavioural rhythms are generated. In the fruit fly (Drosophila melanogaster), the central clock comprises a network of approximately 150 clock neurons, which is important for the maintenance of a coherent and robust rhythm. Several neuropeptides involved in the network have been identified, including Pigment-dispersing factor (PDF) and CCHamide1 (CCHa1) neuropeptides. PDF signals bidirectionally to CCHa1-positive clock neurons; thus, the clock neuron groups expressing PDF and CCHa1 interact reciprocally. However, the role of these interactions in molecular and behavioural rhythms remains elusive. In this study, we generated Pdf (01) and CCHa1(SK8) double mutants and examined their locomotor activity-related rhythms. The single mutants of Pdf (01) or CCHa1(SK8) displayed free-running rhythms under constant dark conditions, whereas approximately 98% of the double mutants were arrhythmic. In light-dark conditions, the evening activity of the double mutants was phase-advanced compared with that of the single mutants. In contrast, both the single and double mutants had diminished morning activity. These results suggest that the effects of the double mutation varied in behavioural parameters. The double and triple mutants of per (01), Pdf (01), and CCHa1(SK8) further revealed that PDF signalling plays a role in the suppression of activity during the daytime under a clock-less background. Our results provide insights into the interactions between PDF and CCHa1 signalling and their roles in activity rhythms.

One of the most important survival strategies of organisms is to avoid predators. Studying one of such strategies, namely, death-feigning behavior, has recently become more common. The success or failure of this antipredator strategy will be affected by the circadian rhythms of both prey and predator because death feigning sometimes has a diurnal rhythm. However, few studies have analyzed the effects of differences in circadian rhythms on predator-avoidance behavior at the genetic level. Recently, the relationship between genes relating to circadian rhythm and death-feigning behavior, an antipredator behavior, has been established at the molecular level. Therefore, in this study, we compared three circadian rhythm-related traits, the free-running period of rhythms, amplitude of circadian rhythms, and total activity of strains of three Tribolium species that were artificially selected for the death-feigning duration: short (S-strains) and long (L-strains) durations. As a result, the amplitude of circadian rhythms and total activity were significantly different between S- and L-strains, but there was no difference in the free-running periods of the rhythm between the strains in T. castaneum, T. confusum, and T. freemani. Although the relationship between death-feigning behavior and activity has been reported for all three species, a genetic relationship between the duration of death feigning and the amplitude of circadian rhythms has been newly found in the present study. It is important to investigate the relationship between antipredator strategies and circadian rhythms at the molecular level in the future.

Animals display a body temperature rhythm (BTR). Little is known about the mechanisms by which a rhythmic pattern of BTR is regulated and how body temperature is set at different times of the day. As small ectotherms, Drosophila exhibit a daily temperature preference rhythm (TPR), which generates BTR. Here, we demonstrate dorsal clock networks that play essential roles in TPR. Dorsal neurons 2 (DN2s) are the main clock for TPR. We find that DN2s and posterior DN1s (DN1ps) contact and the extent of contacts increases during the day and that the silencing of DN2s or DN1ps leads to a lower temperature preference. The data suggest that temporal control of the microcircuit from DN2s to DN1ps contributes to TPR regulation. We also identify anterior DN1s (DN1as) as another important clock for TPR. Thus, we show that the DN networks predominantly control TPR and determine both a rhythmic pattern and preferred temperatures.

Many species show rhythmicity in activity, from the timing of flowering in plants to that of foraging behavior in animals. The free-running periods and amplitude (sometimes called strength or power) of circadian rhythms are often used as indicators of biological clocks. Many reports have shown that these traits are highly geographically variable, and interestingly, they often show latitudinal or longitudinal clines. In many cases, the higher the latitude is, the longer the free-running circadian period (i.e., period of rhythm) in insects and plants. However, reports of positive correlations between latitude or longitude and circadian rhythm traits, including free-running periods, the power of the rhythm and locomotor activity, are limited to certain taxonomic groups. Therefore, we collected a cosmopolitan stored-product pest species, the red flour beetle Tribolium castaneum, in various parts of Japan and examined its rhythm traits, including the power and period of the rhythm, which were calculated from locomotor activity. The analysis revealed that the power was significantly lower for beetles collected in northern areas than southern areas in Japan. However, it is worth noting that the period of circadian rhythm did not show any clines; specifically, it did not vary among the sampling sites, despite the very large sample size (n = 1585). We discuss why these cline trends were observed in T. castaneum.

Circadian clocks prepare the organism to cyclic environmental changes in light, temperature, or food availability. Here, we characterized the master clock in the brain of a strongly photoperiodic insect, the aphid Acyrthosiphon pisum, immunohistochemically with antibodies against A. pisum Period (PER), Drosophila melanogaster Cryptochrome (CRY1), and crab Pigment-Dispersing Hormone (PDH). The latter antibody detects all so far known PDHs and PDFs (Pigment-Dispersing Factors), which play a dominant role in the circadian system of many arthropods. We found that, under long days, PER and CRY are expressed in a rhythmic manner in three regions of the brain: the dorsal and lateral protocerebrum and the lamina. No staining was detected with anti-PDH, suggesting that aphids lack PDF. All the CRY1-positive cells co-expressed PER and showed daily PER/CRY1 oscillations of high amplitude, while the PER oscillations of the CRY1-negative PER neurons were of considerable lower amplitude. The CRY1 oscillations were highly synchronous in all neurons, suggesting that aphid CRY1, similarly to Drosophila CRY1, is light sensitive and its oscillations are synchronized by light-dark cycles. Nevertheless, in contrast to Drosophila CRY1, aphid CRY1 was not degraded by light, but steadily increased during the day and decreased during the night. PER was always located in the nuclei of the clock neurons, while CRY was predominantly cytoplasmic and revealed the projections of the PER/CRY1-positive neurons. We traced the PER/CRY1-positive neurons through the aphid protocerebrum discovering striking similarities with the circadian clock of D. melanogaster: The CRY1 fibers innervate the dorsal and lateral protocerebrum and putatively connect the different PER-positive neurons with each other. They also run toward the pars intercerebralis, which controls hormone release via the neurohemal organ, the corpora cardiaca. In contrast to Drosophila, the CRY1-positive fibers additionally travel directly

The clock network of Drosophila melanogaster expresses various neuropeptides, but a function in clock-mediated behavioral control was so far only found for the neuropeptide pigment dispersing factor (PDF). Here, we propose a role in the control of behavioral rhythms for the ion transport peptide (ITP), which is expressed in the fifth small ventral lateral neuron, one dorsal lateral neuron, and in only a few nonclock cells in the brain. Immunocytochemical analyses revealed that ITP, like PDF, is most probably released in a rhythmic manner at projection terminals in the dorsal protocerebrum. This rhythm continues under constant dark conditions, indicating that ITP release is clock controlled. ITP expression is reduced in the hypomorph mutant ClkAR, suggesting that ITP expression is regulated by CLOCK. Using a genetically encoded RNAi construct, we knocked down ITP in the two clock cells and found that these flies show reduced evening activity and increased nocturnal activity. Overexpression of ITP with two independent timeless-GAL4 lines completely disrupted behavioral rhythms, but only slightly dampened PER cycling in important pacemaker neurons, suggesting a role for ITP in clock output pathways rather than in the communication within the clock network. Simultaneous knockdown (KD) of ITP and PDF made the flies hyperactive and almost completely arrhythmic under constant conditions. Under light-dark conditions, the double-KD combined the behavioral characteristics of the single-KD flies. In addition, it reduced the flies' sleep. We conclude that ITP and PDF are the clock's main output signals that cooperate in controlling the flies' activity rhythms.

Comparative studies on cellular and molecular clock mechanisms have revealed striking similarities in the organization of the clocks among different animal groups. To gain evolutionary insight into the properties of the clock network within the Drosophila genus, we analyzed sequence identities and similarities of clock protein homologues and immunostained brains of 10 different Drosophila species using antibodies against vrille (VRI), PAR-protein domain1 (PDP1), and cryptochrome (CRY). We found that the clock network of both subgenera Sophophora and Drosophila consists of all lateral and dorsal clock neuron clusters that were previously described in Drosophila melanogaster. Immunostaining against CRY and the neuropeptide pigment-dispersing factor (PDF), however, revealed species-specific differences. All species of the Drosophila subgenus and D. pseudoobscura of the Sophophora subgenus completely lacked CRY in the large ventrolateral clock neurons (lLNvs) and showed reduced PDF immunostaining in the small ventrolateral clock neurons (sLNvs). In contrast, we found the expression of the ion transport peptide (ITP) to be consistent within the fifth sLNv and one dorsolateral clock neuron (LNd) in all investigated species, suggesting a conserved putative function of this neuropeptide in the clock. We conclude that the general anatomy of the clock network is highly conserved throughout the Drosophila genus, although there is variation in PDF and CRY expression. Our comparative study is a first step toward understanding the organization of the circadian clock in Drosophila species adapted to different habitats., AuthorCount:7

GABAergic signalling is important for normal sleep in humans and flies. Here we advance the current understanding of GABAergic modulation of daily sleep patterns by focusing on the role of slow metabotropic GABA(B) receptors in the fruit fly Drosophila melanogaster. We asked whether GABA(B)-R2 receptors are regulatory elements in sleep regulation in addition to the already identified fast ionotropic Rdl GABA(A) receptors. By immunocytochemical and reporter-based techniques we show that the pigment dispersing factor (PDF)-positive ventrolateral clock neurons (LNv) express GABA(B)-R2 receptors. Downregulation of GABA(B)-R2 receptors in the large PDF neurons (l-LNv) by RNAi reduced sleep maintenance in the second half of the night, whereas sleep latency at the beginning of the night that was previously shown to depend on ionotropic Rdl GABA(A) receptors remained unaltered. Our results confirm the role of the l-LNv neurons as an important part of the sleep circuit in D. melanogaster and also identify the GABA(B)-R2 receptors as the thus far missing component in GABA-signalling that is essential for sleep maintenance. Despite the significant effects on sleep, we did not observe any changes in circadian behaviour in flies with downregulated GABA(B)-R2 receptors, indicating that the regulation of sleep maintenance via l-LNv neurons is independent of their function in the circadian clock circuit., AuthorCount:6

Animals generally exhibit circadian rhythms of locomotor activity. They initiate locomotor behavior not only reflexively in response to external stimuli but also spontaneously in the absence of any specific stimulus. The neuronal mechanisms underlying circadian locomotor activity can, therefore, be based on the rhythmic changes in either reflexive efficacy or endogenous activity. In crayfish Procambarus clarkii, it can be determined by analyzing electromyographic (EMG) patterns of walking legs whether the walking behavior is initiated reflexively or spontaneously. In this study, we examined quantitatively the leg muscle activity that underlies the locomotor behavior showing circadian rhythms in crayfish. We newly developed a chronic EMG recording system that allowed the animal to freely behave under a tethered condition for more than 10 days. In the LD condition in which the animals exhibited LD entrainment, the rhythmic burst activity of leg muscles for stepping behavior was preceded by non-rhythmic tonic activation that lasted for 1323 +/- 488 ms when the animal initiated walking. In DD and LL free-running conditions, the pre-burst activation lasted for 1779 +/- 31 and 1517 +/- 39 ms respectively. In the mechanical stimulus-evoked walking, the pre-burst activation ended within 79 +/- 6 ms. These data suggest that periodic changes in the crayfish locomotor activity under the condition of LD entrainment or free-running are based on activity changes in the spontaneous initiation mechanism of walking behavior rather than those in the sensori-motor pathway connecting mechanoreceptors with leg movements. (c) 2013 Elsevier B.V. All rights reserved.

Animals generally exhibit circadian rhythms of locomotor activity. They initiate locomotor behavior not only reflexively in response to external stimuli but also spontaneously in the absence of any specific stimulus. The neuronal mechanisms underlying circadian locomotor activity can, therefore, be based on the rhythmic changes in either reflexive efficacy or endogenous activity. In crayfish Procambarus clarkii, it can be determined by analyzing electromyographic (EMG) patterns of walking legs whether the walking behavior is initiated reflexively or spontaneously. In this study, we examined quantitatively the leg muscle activity that underlies the locomotor behavior showing circadian rhythms in crayfish. We newly developed a chronic EMG recording system that allowed the animal to freely behave under a tethered condition for more than 10 days. In the LD condition in which the animals exhibited LD entrainment, the rhythmic burst activity of leg muscles for stepping behavior was preceded by non-rhythmic tonic activation that lasted for 1323 +/- 488 ms when the animal initiated walking. In DD and LL free-running conditions, the pre-burst activation lasted for 1779 +/- 31 and 1517 +/- 39 ms respectively. In the mechanical stimulus-evoked walking, the pre-burst activation ended within 79 +/- 6 ms. These data suggest that periodic changes in the crayfish locomotor activity under the condition of LD entrainment or free-running are based on activity changes in the spontaneous initiation mechanism of walking behavior rather than those in the sensori-motor pathway connecting mechanoreceptors with leg movements. (c) 2013 Elsevier B.V. All rights reserved.

We identified previously two long (DrmITPL1 and -L2) and one amidated short isoform (DrmITP) of insect ion transport peptides (ITPs) as products derived by alternatively splicing from the Drosophila itp-gene (CG13586). The peptides are members of a large family of arthropod neuropeptides incl. crustacean hyperglycemic hormones (CHH/ITP-family), but similar ITPs are only known in locusts to have antidiuretic bioactivity on the hindgut. We localised the peptides by in situ hybridisation and immunocytochemistry with isoform-specific antibodies in the nervous system of larval (L3) and adult Drosophila melanogaster and screened Gal4-lines specific for peptidergic cells. Four neurosecretory cells in brain-corpora cardiaca/allata putatively release DrmITP as a hormone in all stages. DrmITP also occurs in interneurons in the brain/ventral ganglia and in neurons efferent towards the hindgut. Some interneurons are identical to well-known circadian clock neurons for which the effector molecules were elusive but are responsible for the evening bouts of locomotor activity in flies. DrmITPL1 and -L2 were found only in peripheral lateral bipolar and putative sensory neurons which are likely to play a role in the control of growth, hindgut ion transport and heart beat. With DrmITP identified in brain neurosecretory cells, hindgut-innervating neurons in the abdominal ganglia and one pair in the abdomen close to the larval anal organ or innervating the adult rectal pads, both chloride-transporting organs, we are facing an enormous complexity in multiple functions of differentially expressed ITP/Ls derived from a single gene. Preliminary results using Gal4-driven RNAi in distinct peptidergic neurons look promising to find deficiency phenotypes.