Photoperiodic Induction of Diapause Requires Regulated Transcription of timeless in the Larval Brain of Chymomyza costata

Institute of Entomology, Biology Centre AS CR, Ceske Budejovice, Czech Republic.
Journal of Biological Rhythms (Impact Factor: 2.77). 05/2008; 23(2):129-39. DOI: 10.1177/0748730407313364
Source: PubMed


Photoperiodic signal stimulates induction of larval diapause in Chymomyza costata. Larvae of NPD strain (npd-mutants) do not respond to photoperiod. Our previous results indicated that the locus npd could code for the timeless gene and its product might represent a molecular link between circadian and photoperiodic clock systems. Here we present results of tim mRNA (real time-PCR) and TIM protein (immunohistochemistry) analyses in the larval brain. TIM protein was localized in 2 neurons of each brain hemisphere of the 4-d-old 3rd instar wild-type larvae. In a marked contrast, no TIM neurons were detected in the brain of 4-day-old 3rd instar npd -mutant larvae and the level of tim transcripts was approximately 10-fold lower in the NPD than in the wild-type strain. Daily changes in tim expression and TIM presence appeared to be under photoperiodic control in the wild-type larvae. Clear daily oscillations of tim transcription were observed during the development of 3rd instars under the short-day conditions. Daily oscillations were less apparent under the long-day conditions, where a gradual increase of tim transcript abundance appeared as a prevailing trend. Analysis of the genomic structure of tim gene revealed that npd-mutants carry a 1855 bp-long deletion in the 5'-UTR region. This deletion removed the start of transcription and promoter regulatory motifs E-box and TER-box. The authors hypothesize that this mutation was responsible for dramatic reduction of tim transcription rates, disruption of circadian clock function, and disruption of photoperiodic calendar function in npd-mutant larvae of C. costata.

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    • "However, there are some evidence suggesting the connection between the circadian clock system and diapause. For example, the mutation in the promoter region of timeless in the Drosophilid fly, Chymomyza costata, is responsible for the inability of the mutant strain to diapause [35], [36]. Similary, in Drosophila triauraria, allelic differences in two circadian clock genes (timeless and cytochrome) in different strains affected differently on the incidence of diapause [37]. "
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    ABSTRACT: Since its discovery in 1923, the biology of photoperiodism remains a mystery in many ways. We sought the link connecting the circadian system to an endocrine switch, using Antheraea pernyi. PER-, CLK- and CYC-ir were co-expressed in two pairs of dorsolateral neurons of the protocerebrum, suggesting that these are the circadian neurons that also express melatonin-, NAT- and HIOMT-ir. The results suggest that a melatonin pathway is present in the circadian neurons. Melatonin receptor (MT2 or MEL-1B-R)-ir in PTTH-ir neurons juxtaposing clock neurons suggests that melatonin gates PTTH release. RIA showed a melatonin rhythm with a peak four hours after lights off in adult brain both under LD16∶8 (LD) and LD12∶12 (SD), and both the peak and the baseline levels were higher under LD than SD, suggesting a photoperiodic influence. When pupae in diapause were exposed to 10 cycles of LD, or stored at 4°C for 4 months under constant darkness, an increase of NAT activity was observed when PTTH released ecdysone. DNA sequence upstream of nat contained E-boxes to which CYC/CLK could bind, and nat transcription was turned off by clk or cyc dsRNA. dsRNANAT caused dysfunction of photoperiodism. dsRNAPER upregulated nat transcription as anticipated, based on findings in the Drosophila melanogaster circadian system. Transcription of nat, cyc and clk peaked at ZT12. RIA showed that dsRNANAT decreased melatonin while dsRNAPER increased melatonin. Thus nat, a clock controlled gene, is the critical link between the circadian clock and endocrine switch. MT-binding may release PTTH, resulting in termination of diapause. This study thus examined all of the basic functional units from the clock: a photoperiodic counter as an accumulator of mRNANAT, to endocrine switch for photoperiodism in A. pernyi showing this system is self-complete without additional device especially for photoperiodism.
    Full-text · Article · Mar 2014 · PLoS ONE
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    • "In fact, the mRNA of per and tim of the head sample reach their peaks in abundance 4 h after “light-off” during photoperiods of 16-h light and 8-h darkness (LD, 16:8), LD 12:12, and LD 8:16, and oscillation uses “lights-off” as a phase reference point (Qiu and Hardin, 1996). Setting peaks of per or tim mRNA levels at “lights-off” have also been observed in the head of the flesh fly Sarcophaga crassipalpis, the whole central nervous system of C. costata, and the brain of P. terraenovae (Goto and Denlinger, 2002; Stehlík et al., 2008; Muguruma et al., 2010). Other types of responses of circadian clock gene expression to photoperiods have also been observed. "
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    ABSTRACT: Since Bünning's observation of circadian rhythms and photoperiodism in the runner bean Phaseolus multiflorus in 1936, many studies have shown that photoperiodism is based on the circadian clock system. In insects, involvement of circadian clock genes or neurons has been recently shown in the photoperiodic control of developmental arrests, diapause. Photoperiod sets peaks of period (per) or timeless (tim) mRNA abundance at lights-off in Sarcophaga crassipalpis, Chymomyza costata and Protophormia terraenovae. Abundance of per and Clock mRNA changes by photoperiod in Pyrrhocoris apterus. Subcellular Per distribution in circadian clock neurons changes with photoperiod in P. terraenovae. Although photoperiodism is not known in Leucophaea maderae, under longer day length, more stomata and longer commissural fibers of circadian clock neurons have been found. These plastic changes in the circadian clock neurons could be an important constituent for photoperiodic clock mechanisms to integrate repetitive photoperiodic information and produce different outputs based on day length.
    Full-text · Article · Aug 2013 · Frontiers in Physiology
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    • "The genetic basis of dormancy has been partially explained in insect species (Emerson et al. 2009). For example, the analysis of photoperiodic mutants in the fly Chymomyza costata and the linden bug Pyrrhocoris apterus has revealed that the alteration of circadian clock gene expression is necessary to enter dormancy (Syrov a et al. 2003; Stehlik et al. 2008), while complex gene cascades and hormone interactions are responsible for maintaining and ending it. (Emerson et al. 2009; Tapia & Morano 2010; for review see Denlinger 2002). "
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