The role of circadian clock genes in the photoperiodic timer of the linden bug Pyrrhocoris apterus during the nymphal stage

Many insects survive seasonal adversities during diapause, a form of programmed developmental and metabolic arrest. Photoperiodically regulated entry into diapause allows multivoltine insect species to optimize the number of generations. The molecular mechanism of the photoperiodic timer is unknown in insects. In the present study, we take advantage of the robust reproductive diapause response in the linden bug Pyrrhocoris apterus and explore the fifth‐instar nymphal stage, which is the most photoperiod‐sensitive stage. The nymphs display daily changes in locomotor activity during short days; this differs from the activity observed during long days. We find evidence of cyclical expression of the circadian clock genes, per and cyc, in nymphal heads; in addition, per expression is also photoperiod‐dependent. The RNA interference‐mediated knockdown of the two circadian clock genes, Clk and cyc, during the nymphal stage results in reproductive arrest in adult females. Furthermore, Clk and cyc knockdown induces the expression of the storage protein hexamerin in the fat body, whereas the expression of vitellogenin diminishes. Taken together, these data support the involvement of circadian clock genes in photoperiodic timer and/or diapause induction.     

Association between circadian clock genes and diapause incidence in Drosophila triauraria

Diapause is an adaptive response triggered by seasonal photoperiodicity to overcome unfavorable seasons. The photoperiodic clock is a system that controls seasonal physiological processes, but our knowledge about its physiological mechanisms and genetic architecture remains incomplete. The circadian clock is another system that controls daily rhythmic physiological phenomena. It has been argued that there is a connection between the two clocks. To examine the genetic connection between them, we analyzed the associations of five circadian clock genes (period, timeless, Clock, cycle and cryptochrome) with the occurrence of diapause in Drosophila triauraria, which shows a robust reproductive diapause with clear photoperiodicity. Non-diapause strains found in low latitudes were compared in genetic crosses with the diapause strain, in which the diapause trait is clearly dominant. Single nucleotide polymorphism and deletion analyses of the five circadian clock genes in backcross progeny revealed that allelic differences in timeless and cryptochrome between the strains were additively associated with the differences in the incidence of diapause. This suggests that there is a molecular link between certain circadian clock genes and the occurrence of diapause.     

Deleting the Arntl clock gene in the granular layer of the mouse cerebellum: impact on the molecular circadian clockwork

The suprachiasmatic nucleus houses the central circadian clock and is characterized by the timely regulated expression of clock genes. However, neurons of the cerebellar cortex also contain a circadian oscillator with circadian expression of clock genes being controlled by the suprachiasmatic nucleus. It has been suggested that the cerebellar circadian oscillator is involved in food anticipation, but direct molecular evidence of the role of the circadian oscillator of the cerebellar cortex is currently unavailable. To investigate the hypothesis that the circadian oscillator of the cerebellum is involved in circadian physiology and food anticipation, we therefore by use of Cre‐LoxP technology generated a conditional knockout mouse with the core clock gene Arntl deleted specifically in granule cells of the cerebellum, since expression of clock genes in the cerebellar cortex is mainly located in this cell type. We here report that deletion of Arntl heavily influences the molecular clock of the cerebellar cortex with significantly altered and arrhythmic expression of other central clock and clock‐controlled genes. On the other hand, daily expression of clock genes in the suprachiasmatic nucleus was unaffected. Telemetric registrations in different light regimes did not detect significant differences in circadian rhythms of running activity and body temperature between Arntl conditional knockout mice and controls. Furthermore, food anticipatory behavior did not differ between genotypes. These data suggest that Arntl is an essential part of the cerebellar oscillator; however, the oscillator of the granular layer of the cerebellar cortex does not control traditional circadian parameters or food anticipation.     

Explaining the sawtooth: latitudinal periodicity in a circadian gene correlates with shifts in generation number

Many temperate insects take advantage of longer growing seasons at lower latitudes by increasing their generation number or voltinism. In some insects, development time abruptly decreases when additional generations are fit into the season. Consequently, latitudinal ‘sawtooth’ clines associated with shifts in voltinism are seen for phenotypes correlated with development time, like body size. However, latitudinal variation in voltinism has not been linked to genetic variation at specific loci. Here, we show a pattern in allele frequency among voltinism ecotypes of the European corn borer moth (Ostrinia nubilalis) that is reminiscent of a sawtooth cline. We characterized 145 autosomal and sex‐linked SNPs and found that period, a circadian gene that is genetically linked to a major QTL determining variation in post‐diapause development time, shows cyclical variation between voltinism ecotypes. Allele frequencies at an unlinked circadian clock gene cryptochrome1 were correlated with period. These results suggest that selection on development time to ‘fit’ complete life cycles into a latitudinally varying growing season produces oscillations in alleles associated with voltinism, primarily through changes at loci underlying the duration of transitions between diapause and other life history phases. Correlations among clock loci suggest possible coupling between the circadian clock and the circannual rhythms for synchronizing seasonal life history. We anticipate that latitudinal oscillations in allele frequency will represent signatures of adaptation to seasonal environments in other insects and may be critical to understanding the ecological and evolutionary consequences of variable environments, including response to global climate change.     

Diapause induction and photoperiodic clock in Chilo suppressalis (Walker) (Lepidoptera: Crambidae)

The mature larvae of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Crambidae) enters facultative diapause in response to short‐day conditions in the autumn (August–September). Diapause induction and photoperiodic clock mechanism were investigated in C. suppressalis larvae reared on an artificial diet in the present study. The critical night length for diapause induction was about 9 h 53 min to 10 h 39 min at 22 to 28°C. The third‐instar larvae were found to be relatively sensitive to diapause induction. Photoperiodic response under non‐24‐h light–dark cycles showed that scotophase length played an essential role in the induction of larval diapause in C. suppressalis, and consecutive exposure to long‐night cycles was necessary for a high diapause incidence. In the Nanda–Hamner experiment, diapause incidence peaked at scotophase of 12 h and dropped rapidly at scotophases > 24 h. In the Bünsow experiment, diapause incidence was clearly suppressed, especially at the light pulse located 8 h in the scotophase. Both the Nanda–Hamner and Bünsow experiments showed no rhythmic fluctuations with a period of about 24 h; thus the photoperiodic clock in C. suppressalis is a non‐oscillatory hourglass timer or a rapidly damping circadian oscillator.     

The metabolic sensor AKIN10 modulates the Arabidopsis circadian clock in a light‐dependent manner

Plants generate rhythmic metabolism during the repetitive day/night cycle. The circadian clock produces internal biological rhythms to synchronize numerous metabolic processes such that they occur at the required time of day. Metabolism conversely influences clock function by controlling circadian period and phase and the expression of core‐clock genes. Here, we show that AKIN10, a catalytic subunit of the evolutionarily conserved key energy sensor sucrose non‐fermenting 1 (Snf1)‐related kinase 1 (SnRK1) complex, plays an important role in the circadian clock. Elevated AKIN10 expression led to delayed peak expression of the circadian clock evening‐element GIGANTEA (GI) under diurnal conditions. Moreover, it lengthened clock period specifically under light conditions. Genetic analysis showed that the clock regulator TIME FOR COFFEE (TIC) is required for this effect of AKIN10. Taken together, we propose that AKIN10 conditionally works in a circadian clock input pathway to the circadian oscillator.     

Mendelian inheritance of pupal diapause in the flesh fly, Sarcophaga bullata

Pupal diapause (dormancy) in the flesh fly, Sarcophaga bullata, is induced by short-day photoperiods and low temperature. In this study, the inheritance mode of diapause was investigated by crossing a nondiapausing (nd) strain of S. bullata with 2 diapausing strains having different diapause capacities. The results consistently indicated that diapause incidence is inherited in a simple Mendelian pattern, thus a single gene or a small gene cluster linked to the photoperiodic clock controls the seasonal response of diapause. The fact that the nd strain lacked daily rhythmicity in adult eclosion and showed altered expression of 2 circadian clock genes suggests that the photoperiodic and circadian clocks are related through a shared molecular component in S. bullata.     

Circadian clock genes period and cycle regulate photoperiodic diapause in the bean bug Riptortus pedestris males

The photoperiodic response is crucial for many insects to adapt to seasonal changes in temperate regions. It was recently shown that the circadian clock genes period (per) and cycle (cyc) are involved in the photoperiodic regulation of reproductive diapause in the bean bug Riptortus pedestris females. Here, we investigated the involvement of per and cyc both in the circadian rhythm of cuticle deposition and in the photoperiodic diapause of R. pedestris males using RNA interference (RNAi). RNAi of per and cyc disrupted the cuticle deposition rhythm and resulted in distinct cuticle layers. RNAi of per induced development of the male reproductive organs even under diapause-inducing short-day conditions, whereas RNAi of cyc suppressed development of the reproductive organs even under diapause-averting long-day conditions. Thus, the present study suggests that the circadian clock operated by per and cyc governs photoperiodism of males as that of females.     

Circadian clock regulates photoperiodic responses governed by distinct output pathways in the bean bug,Riptortus pedestris

Effects of RNA interference (RNAi) targeted against circadian clock genes on two distinct types of photoperiodic responses – ovarian development and lipid accumulation – were investigated in a bean bug Riptortus pedestris, to explore which physiological process in the photoperiodic response involved the circadian clock. Ovarian development and lipid accumulation are known to be regulated by distinct output pathways. Control insects showed clear photoperiodic responses; i.e. induction of ovarian development and suppression of lipid accumulation under long-day conditions, whereas opposite characteristics under short-day conditions. We found that RNAi directed against period, a negative element of the circadian clock, produced a long-day effect for both the ovarian development and lipid accumulation, while RNAi directed against Clock, a positive element of the circadian clock, produced a short-day effect for both, irrespective of photoperiod. These results indicate that the circadian clock comprised of these genes regulates a process governing both distinct photoperiodic responses.     

A nondiapausing variant of the flesh fly, Sarcophaga bullata, that shows arrhythmic adult eclosion and elevated expression of two circadian clock genes, period and timeless

We describe a variant of the flesh fly, Sarcophaga bullata, which fails to enter pupal diapause in response to short daylength and low temperatures. This fly also has an arrhythmic adult eclosion pattern: rather than eclosing in early photophase, the variant ecloses arrhythmically throughout the photophase and scotophase. The loss of both diapause (photoperiodic response) and the gating of adult eclosion (presumably a circadian response) suggests that the same clock system is involved in these two responses. An examination of the expression patterns of the clock genes period and timeless demonstrates that both genes are present in the nondiapausing variant, but surprisingly, both genes are expressed at higher levels. This abnormality we observe, possibly the consequence of an upstream clock gene malfunction or a malfunction of the autoregulatory loop, results in disruption of a component of the clock system that is apparently needed for both photoperiodism and circadian rhythmicity.     

Light- and temperature-entrainable circadian clock in soybean development

In plants, the spatiotemporal expression of circadian oscillators provides adaptive advantages in diverse species. However, the molecular basis of circadian clock in soybean is not known. In this study, we used soybean hairy roots expression system to monitor endogenous circadian rhythms and the sensitivity of circadian clock to environmental stimuli. We discovered in experiments with constant light and temperature conditions that the promoters of clock genes GmLCLb2 and GmPRR9b1 drive a self-sustained, robust oscillation of about 24-h in soybean hairy roots. Moreover, we demonstrate that circadian clock is entrainable by ambient light/dark or temperature cycles. Specifically, we show that light and cold temperature pulses can induce phase shifts of circadian rhythm, and we found that the magnitude and direction of phase responses depends on the specific time of these two zeitgeber stimuli. We obtained a quadruple mutant lacking the soybean gene GmLCLa1, LCLa2, LCLb1, and LCLb2 using CRISPR, and found that loss-of-function of these four GmLCL orthologs leads to an extreme short-period circadian rhythm and late-flowering phenotype in transgenic soybean. Our study establishes that the morning-phased GmLCLs genes act constitutively to maintain circadian rhythmicity and demonstrates that their absence delays the transition from vegetative growth to reproductive development.     

Interaction of MAGED1 with nuclear receptors affects circadian clock function

The circadian clock has a central role in physiological adaption and anticipation of day/night changes. In a genetic screen for novel regulators of circadian rhythms, we found that mice lacking MAGED1 (Melanoma Antigen Family D1) exhibit a shortened period and altered rest–activity bouts. These circadian phenotypes are proposed to be caused by a direct effect on the core molecular clock network that reduces the robustness of the circadian clock. We provide in vitro and in vivo evidence indicating that MAGED1 binds to RORα to bring about positive and negative effects on core clock genes of Bmal1, Rev‐erbα and E4bp4 expression through the Rev‐Erbα/ROR responsive elements (RORE). Maged1 is a non‐rhythmic gene that, by binding RORα in non‐circadian way, enhances rhythmic input and buffers the circadian system from irrelevant, perturbing stimuli or noise. We have thus identified and defined a novel circadian regulator, Maged1, which is indispensable for the robustness of the circadian clock to better serve the organism.     

Light induction of the clock-controlled geneccg-1 is not transduced through the circadian clock inNeurospora crassa

Ambient light and the circadian clock have been shown to be capable of acting either independently or in an interrelated fashion to regulate the expression of conidiation in the ascomycete fungusNeurospora crassa. Recently several molecular correlates of the circadian clock have been identified in the form of the morning-specific clock-controlled genesccg-1 andccg-2. In this paper we report studies on the regulation ofccg-1, an abundantly expressed gene displaying complex regulation. Consistent with an emerging consensus for clock-controlled genes and conidiation genes inNeurospora, we report thatccg-1 expression is induced by light, and show that this induction is independent of the direct effects of light on the circadian clock. Although circadian regulation of the gene is lost in strains lacking a functional clock, expression ofccg-1 is still not constitutive, but rather fluctuates in concert with changes in developmental potential seen in such strains. Light induction ofccg-1 requires the products of theNeurospora wc-1 andwc-2 genes, but surprisingly the requirement forwc-2 is suppressed in conditional mutants ofcot-1, a gene that encodes a cAMP-dependent protein kinase. These data provide insight into a complex regulatory web, involving at least circadian clock control, light control, metabolic control, and very probably developmental regulation, that governs the expression ofccg-1.