Behavioral Dissection of the Drosophila Circadian Multioscillator System that Regulates Locomotor Rhythms

The fruit fly, Drosophila melanogaster, shows a bimodal circadian activity rhythm with peaks around light-on and before light-off. This rhythm is driven by seven groups of so-called clock neurons in the brain. To dissect the multioscillatory nature of the Drosophila clock system, the process of reentrainment to a reversed light cycle was examined by using wild-type flies and cry(b) mutant flies that carry a strong loss-of-function mutation in cryptochrome (cry) gene. The wild-type flies showed that the morning peak dissociated into two components, while a substantial fraction of cry(b) flies exhibited dissociation of the evening peak into two components that shifted in different directions. When the temperature cycle was given in constant darkness in such a manner that the thermophase corresponded to the previous night phase, the morning peak also split into two components in wild-type flies. These results suggest that both morning and evening peaks are driven by two separate oscillators that have different entrainability to light and temperature cycles. Examination of the process of reentrainment to a reversed LD in mutant flies that lack some of the four known circadian photoreceptors (compound eyes, ocelli, CRYPTOCHROME [CRY], and Hofbauer-Buchner [H-B] eyelets) revealed that these four photoreceptors play different roles in photic entrainment of the four putative oscillators

Phase Shifts of the Circadian Locomotor Rhythm Induced by Pigment-Dispersing Factor in the Cricket Gryllus bimaculatus

Pigment-dispersing factors (PDFs) are octadeca-peptides widely distributed in insect optic lobes and brain. In this study, we have purified PDF and determined its amino acid sequence in the cricket Gryllus bimaculatus. Its primary structure was NSEIINSLLGLPKVLNDA-NH2, homologous to other PDH family members so far reported. When injected into the optic lobe of experimentally blinded adult male crickets, Gryllus-PDF induced phase shifts in their activity rhythms in a phase dependent and dose dependent manner. The resulted phase response curve (PRC) showed delays during the late subjective night to early subjective day and advances during the mid subjective day to mid subjective night. The PRC was different in shape from those for light, serotonin and temperature. These results suggest that PDF plays a role in phase regulation of the circadian clock through a separate pathway from those of other known phase regulating agents

Analyses of the circadian clock using a cricket as an experimental animal: Functional analysis of the clock gene period in circadian rhythm generation

フタホシコオロギでは､分子生物学的手法が開発されつつある｡ここでは､最近われわれが試みているRNA干渉による､フタホシコオロギ(Gyyllus bimaculatus)の時計遺伝子perの機能解析を紹介した。幼虫頭部での解析により､per mRNAの発現量は､夜の始めにピークをもつリズムを示し､このリズムは恒暗･恒温条件下でも継続することが明らかとなり､perの時計機構への関与が示唆された。そこで､per dsRNAを用いたRNA干渉により､per遺伝子の役割を検討した。幼虫へのper dsRNAの投与により､per mRNAレベルは対照群の25%にまで減少し､かつほとんどの個体で活動リズムが消失することがわかった。これらの結果はコオロギでもperがリズムの発現に重要な役割を担うことを示唆している。さらに､終齢幼虫にper dsRNAを投与した場合も､羽化後の活動が恒暗条件下で無周期となることがわかった。これらの結果から､コオロギではperが時計機構に必須であることが示唆された。また同時に､RNA干渉が時計遺伝子の機能解析に極めて有効な手段であることが確認された

The Nuclear Receptor Genes HR3 and E75 Are Required for the Circadian Rhythm in a Primitive Insect

Insect circadian rhythms are generated by a circadian clock consisting of transcriptional/translational feedback loops, in which CYCLE and CLOCK are the key elements in activating the transcription of various clock genes such as timeless (tim) and period (per). Although the transcriptional regulation of Clock (Clk) has been profoundly studied, little is known about the regulation of cycle (cyc). Here, we identify the orphan nuclear receptor genes HR3 and E75, which are orthologs of mammalian clock genes, Rorα and Rev-erbα, respectively, as factors involved in the rhythmic expression of the cyc gene in a primitive insect, the firebrat Thermobia domestica. Our results show that HR3 and E75 are rhythmically expressed, and their normal, rhythmic expression is required for the persistence of locomotor rhythms. Their RNAi considerably altered the rhythmic transcription of not only cyc but also tim. Surprisingly, the RNAi of HR3 revealed the rhythmic expression of Clk, suggesting that this ancestral insect species possesses the mechanisms for rhythmic expression of both cyc and Clk genes. When either HR3 or E75 was knocked down, tim, cyc, and Clk or tim and cyc, respectively, oscillated in phase, suggesting that the two genes play an important role in the regulation of the phase relationship among the clock genes. Interestingly, HR3 and E75 were also found to be involved in the regulation of ecdysis, suggesting that they interconnect the circadian clock and developmental processes

Effects of Unilateral Compound-Eye Removal on the Photoperiodic Responses of Nymphal Development in the Cricket Modicogryllus siamensis

The cricket, Modicogryllus siamensis, shows clear photoperiodic responses at 25 degrees C in nymphal development. Under long-day conditions (LD16:8), nymphs became adults about 50 days after hatching, while under short-day conditions (LD8:16) the duration of nymphal stage extended to more than 130 days. Under constant dark conditions, two developmental patterns were observed: about 60% of crickets became adults slightly slower than under the long-day conditions, and the rest at later than 100 days after hatching, like those under the short-day conditions. When the compound eye was unilaterally removed on the 2nd day of hatching, an increase of molting and an extension of the nymphal period were observed under the long-day conditions, while under the short-day conditions, some crickets developed faster and others slower than intact crickets. These results suggest that this cricket receives photoperiodic information through the compound eye, that a pair of the compound eyes is required for a complete photoperiodic response, and that interaction between bilateral circadian clocks may be also involved in the response

Enhancer of zeste plays an important role in photoperiodic modulation of locomotor rhythm in the cricket, Gryllus bimaculatus

Introduction: Insects show daily behavioral rhythms controlled by an endogenous oscillator, the circadian clock. The rhythm synchronizes to daily light–dark cycles (LD) and changes waveform in association with seasonal change in photoperiod. Results: To explore the molecular basis of the photoperiod-dependent changes in circadian locomotor rhythm, we investigated the role of a chromatin modifier, Enhancer of zeste (Gb’E(z)), in the cricket, Gryllus bimaculatus. Under a 12 h:12 h LD (LD 12:12), Gb’E(z) was constitutively expressed in the optic lobe, the site of the biological clock; active phase (α) and rest phase (ρ) were approximately 12 h in duration, and α/ρ ratio was approximately 1.0. When transferred to LD 20:4, the α/ρ ratio decreased significantly, and the Gb’E(z) expression level was significantly reduced at 6 h and 10 h after light-on, as was reflected in the reduced level of trimethylation of histone H3 lysine 27. This change was associated with change in clock gene expression profiles. The photoperiod-dependent changes in α/ρ ratio and clock gene expression profiles were prevented by knocking down Gb’E(z) by RNAi. Conclusions: These results suggest that histone modification by Gb’E(z) is involved in photoperiodic modulation of the G. bimaculatus circadian rhythm

Risk for lung cancer in workers exposed to benzidine and/or beta-naphthylamine: a protocol for systematic review and meta-analysis.

BACKGROUND:Risk for lung cancer in workers exposed to benzidine (BZ) and/or beta-naphthylamine (BNA), which are well-known bladder carcinogens, has been examined in many epidemiological studies, but individual epidemiological studies generally lack the power to examine the association between BZ/BNA exposure and lung cancer. We conduct a systematic review and meta-analysis to determine the risk for lung cancer among workers exposed to BZ/BNA occupationally. METHODS/DESIGN:Studies will be identified by a MEDLINE, EMBASE, CDSR, and CINAHL search and by the reference lists of articles/relevant reviews. Eligible studies will be cohort and case-control studies that report occupational BZ/BNA exposure and the outcome of interest (lung cancer death/incidence). The method of meta-analysis will be used to combine standardized mortality ratios (SMRs) and/or standardized incidence ratios (SIRs) from retrospective and prospective cohort studies and odds ratios (ORs) from case-control studies. Two reviewers will independently screen articles, extract data, and assess scientific quality using standardized forms and published quality assessment tools tailored for each study design. Overall pooled risk estimates and their corresponding 95% confidence intervals (CIs) will be obtained using random effects model. This systematic review and meta-analysis will be conducted following the Meta-analysis of Observational Studies in Epidemiology (MOOSE) guidelines, and results will be reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement.DISCUSSION: This review will identify and synthesize studies of the association between occupational BZ/BNA exposure and lung cancer. The findings will help to identify whether BZ/BNA could cause lung cancer and might indicate whether workers with exposure to BZ/BNA have a need for preventive measures against non-urological cancer besides bladder cancer.© 2014 Tomioka et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,unless otherwise stated

The role of clockwork orange in the circadian clock of the cricket Gryllus bimaculatus

The circadian clock generates rhythms of approximately 24 h through periodic expression of the clock genes. In insects, the major clock genes period (per) and timeless (tim) are rhythmically expressed upon their transactivation by CLOCK/CYCLE, with peak levels in the early night. In Drosophila, clockwork orange (cwo) is known to inhibit the transcription of per and tim during the daytime to enhance the amplitude of the rhythm, but its function in other insects is largely unknown. In this study, we investigated the role of cwo in the clock mechanism of the cricket Gryllus bimaculatus. The results of quantitative RT-PCR showed that under a light/dark (LD) cycle, cwo is rhythmically expressed in the optic lobe (lamina-medulla complex) and peaks during the night. When cwo was knocked down via RNA interference (RNAi), some crickets lost their locomotor rhythm, while others maintained a rhythm but exhibited a longer free-running period under constant darkness (DD). In cwo(RNAi) crickets, all clock genes except for cryptochrome 2 (cry2) showed arrhythmic expression under DD; under LD, some of the clock genes showed higher mRNA levels, and tim showed rhythmic expression with a delayed phase. Based on these results, we propose that cwo plays an important role in the cricket circadian clock

The role of clockwork orange in the circadian clock of the cricket Gryllus bimaculatus

The circadian clock generates rhythms of approximately 24 h through periodic expression of the clock genes. In insects, the major clock genes period (per) and timeless (tim) are rhythmically expressed upon their transactivation by CLOCK/CYCLE, with peak levels in the early night. In Drosophila, clockwork orange (cwo) is known to inhibit the transcription of per and tim during the daytime to enhance the amplitude of the rhythm, but its function in other insects is largely unknown. In this study, we investigated the role of cwo in the clock mechanism of the cricket Gryllus bimaculatus. The results of quantitative RT-PCR showed that under a light/dark (LD) cycle, cwo is rhythmically expressed in the optic lobe (lamina-medulla complex) and peaks during the night. When cwo was knocked down via RNA interference (RNAi), some crickets lost their locomotor rhythm, while others maintained a rhythm but exhibited a longer free-running period under constant darkness (DD). In cwoRNAi crickets, all clock genes except for cryptochrome 2 (cry2) showed arrhythmic expression under DD; under LD, some of the clock genes showed higher mRNA levels, and tim showed rhythmic expression with a delayed phase. Based on these results, we propose that cwo plays an important role in the cricket circadian clock

Cryptochrome-dependent and -independent circadian entrainment circuits in Drosophila.

　Entrainment to environmental light/dark (LD) cycles is a central function of circadian clocks. In Drosophila, entrainment is achieved by Cryptochrome (CRY) and input from the visual system. During activation by brief light pulses, CRY triggers the degradation of TIMELESS and subsequent shift in circadian phase. This is less important for LD entrainment, leading to questions regarding light input circuits and mechanisms from the visual system. Recent studies show that different subsets of brain pacemaker clock neurons, the morning (M) and evening (E) oscillators, have distinct functions in light entrainment. However, the role of CRY in M and E oscillators for entrainment to LD cycles is unknown. Here, we address this question by selectively expressing CRY in different subsets of clock neurons in a cry-null (cry0) mutant background. We were able to rescue the light entrainment deficits of cry0 mutants by expressing CRY in E oscillators but not in any other clock neurons. Par domain protein 1 molecular oscillations in the E, but not M, cells of cry0 mutants still responded to the LD phase delay. This residual light response was stemming from the visual system because it disappeared when all external photoreceptors were ablated genetically. We concluded that the E oscillators are the targets of light input via CRY and the visual system and are required for normal light entrainment