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.     

Night-interruption experiments and action spectra for dawn and dusk in relation to the photoperiodic clock of the cabbage whitefly,Aleyrodes proletella (Homoptera: Aleyrodidae)

The dark period (scotophase) is the most photoperiodically important part of a light-dark cycle in Aleyrodes proletella. Night-interruption studies have revealed three distinct dark stages: the photosensitive stage 1 lasts for about 3 h after dusk and 1-h light breaks both stop and re-set the photoperiodic clock; stage 2 also lasts about 3 h, but is photorefractory to some degree; stage 3 is photosensitive, but short light breaks do not re-set the clock although a 4-h light break (equivalent to a main photophase) does restore the capacity to respond to a normal critical night length in the post-interruption scotophase.Action spectra revealed peak photoperiodic sensitivity to blue light (410–430 nm) with 50% responses., at 1.5 μWcm⁻² and 2.5 μWcm⁻² for the dusk and dawn peaks respectively. These data are consistent with the view that the photopigment is a carotenoprotein.The results are interpreted in terms of the photoperiodic clock in A. proletella operating on the hour glass principle.     

The circadian clock genes affect reproductive capacity in the desert locust Schistocerca gregaria

The circadian clocks govern many metabolic and behavioral processes in an organism. In insects, these clocks and their molecular machinery have been found to influence reproduction in many different ways. Reproductive behavior including courtship, copulation and egg deposition, is under strong influence of the daily rhythm. At the molecular level, the individual clock components also have their role in normal progress of oogenesis and spermatogenesis. In this study on the desert locust Schistocerca gregaria, three circadian clock genes were identified and their expression profiles were determined. High expression was predominantly found in reproductive tissues. Similar daily expression profiles were found for period (per) and timeless (tim), while the clock (clk) mRNA level is higher 12 h before the first per and tim peak. A knockdown of either per or tim resulted in a significant decrease in the progeny produced by dsRNA treated females confirming the role of clock genes in reproduction and providing evidence that both PER and TIM are needed in the ovaries for egg development. Since the knockdown of clk is lethal for the desert locust, its function remains yet to be elucidated.     

Photoperiodism in Neurospora crassa

Plants and animals use day or night length for seasonal control of reproduction and other biological functions. Overwhelming evidence suggests that this photoperiodic mechanism relies on a functional circadian system. Recent progress has defined how flowering time in plants is regulated by photoperiodic control of output pathways, but the underlying mechanisms of photoperiodism remain to be described. The authors investigate photoperiodism in a genetic model system for circadian rhythms research, Neurospora crassa. They find that both propagation and reproduction respond systematically to photoperiod. Furthermore, a nonreproductive light-regulated function is also enhanced under certain photoperiodic conditions. All of these photoperiodic responses require a functional circadian clock, in that they are absent in a clock mutant. Night break experiments show that measuring night length is one of the mechanisms used for photoperiod assessment. This represents the first formal report of photoperiodism in the fungi.     

Photoperiodic time measurement in the spider mite Tetranychus urticae: A novel concept

To explain photoperiodic induction of diapause in the spider mite Tetranychus urticae a new theoretical model was developed which took into account both the hourglass and rhythmic elements shown to be present in the photoperiodic reaction of these mites. It is emphasized that photoperiodic induction is the result of time measurement as well as the summation and integration of a number of successive photoperiodic cycles: the model, therefore, consists of separate ‘clock’ and ‘counter’ mechanisms. In current views involvement of the circadian system in photoperiodism is interpreted in terms of the hypothesis that the photoperiodic clock itself is based on one or more circadian oscillators. Here a different approach has been chosen as regards the role of the circadian system in photoperiodism: the possibility, previously put forward by other authors, that some aspect of the photoperiodic induction mechanism other than the clock is controlled by the circadian system was investigated by assuming a circadian influence on the photoperiodic counter mechanism. The derivation of this ‘hourglass timer oscillator counter’ model of photoperiodic induction in T. urticae is described and its operation demonstrated on the basis of a number of diel and nondiel photoperiods, with and without light interruptions.     

An instantly damping oscillator model for photoperiodic time measurement in the aphid Aphis fabae

It is assumed that a non-repetitive photoperiodic clock, or “hourglass”, could be circadian based, and described as an instantly damping circadian oscillator. A model for an instantly damping oscillator is developed in the present paper and tested on photoperiodic morph determination in the black bean aphid, Aphis fabae. The kinetics of the clock are presented in the form of phase resetting curves which plot the phase of the oscillation at lights-on against the phase at lights-off. Other components of the model, that is a “counter”, that accumulates and integrates photoperiodic information contained in a number of light-dark cycles up to a threshold value for induction to occur, and an influence of the circadian system on the induction process, are as previously described in the “hourglass timer-oscillator counter” model of photoperiodic induction of diapause in the spider mite, Tetranychus urticae. It is shown that night-length measurement in A. fabae can be described by means of an instantly damping oscillator: the phase resetting curves are based on a number of photoperiodic experiments and resemble the phase resetting curves determined for overt circadian rhythms in other insects. However, the results do not distinguish between a photoperiodic clock based on a damped circadian oscillator or a non-circadian hourglass mechanism.     

Deciphering time measurement: the role of circadian 'clock' genes and formal experimentation in insect photoperiodism

This review examines possible role(s) of circadian ‘clock’ genes in insect photoperiodism against a background of many decades of formal experimentation and model building. Since ovarian diapause in the genetic model organism Drosophila melanogaster has proved to be weak and variable, recent attention has been directed to species with more robust photoperiodic responses. However, no obvious consensus on the problem of time measurement in insect photoperiodism has yet to emerge and a variety of mechanisms are indicated. In some species, expression patterns of clock genes and formal experiments based on the canonical properties of the circadian system have suggested that a damped oscillator version of Pittendrigh's external coincidence model is appropriate to explain the measurement of seasonal changes in night length. In other species extreme dampening of constituent oscillators may give rise to apparently hourglass-like photoperiodic responses, and in still others there is evidence for dual oscillator (dawn and dusk) photoperiodic mechanisms of the internal coincidence type. Although the exact role of circadian rhythmicity and of clock genes in photoperiodism is yet to be settled, Bünning's general hypothesis (Bünning, 1936) remains the most persuasive unifying principle. Observed differences between photoperiodic clocks may be reflections of underlying differences in the clock genes in their circadian feedback loops.     

Distribution of PERIOD-immunoreactive neurons and temporal change of the immunoreactivity under long-day and short-day conditions in the larval brain of the flesh fly Sarcophaga similis

The flesh fly Sarcophaga similis show a clear photoperiodic response; they develop into adults under long days, whereas they arrest their development at the pupal stage under short days. Although the involvement of a circadian clock in photoperiodic time measurement is suggested in this species, the anatomical location of the clock neurons responsible for the time measurement has been unknown. We detected two PERIOD-immunoreactive cell clusters in the larval brain; one cluster was located at the dorsoanterior region and the other at the medial region. We further investigated their temporal changes in PERIOD-immunoreactivity and compared their patterns under different photoperiods.     

Drosophila free-running rhythms require intercellular communication

Robust self-sustained oscillations are a ubiquitous characteristic of circadian rhythms. These include Drosophila locomotor activity rhythms, which persist for weeks in constant darkness (DD). Yet the molecular oscillations that underlie circadian rhythms damp rapidly in many Drosophila tissues. Although much progress has been made in understanding the biochemical and cellular basis of circadian rhythms, the mechanisms that underlie the differences between damped and self-sustaining oscillations remain largely unknown. A small cluster of neurons in adult Drosophila brain, the ventral lateral neurons (LN_vs), is essential for self-sustained behavioral rhythms and has been proposed to be the primary pacemaker for locomotor activity rhythms. With an LN_v-specific driver, we restricted functional clocks to these neurons and showed that they are not sufficient to drive circadian locomotor activity rhythms. Also contrary to expectation, we found that all brain clock neurons manifest robust circadian oscillations of timeless and cryptochrome RNA for many days in DD. This persistent molecular rhythm requires pigment-dispersing factor (PDF), an LN_v-specific neuropeptide, because the molecular oscillations are gradually lost when Pdf⁰¹ mutant flies are exposed to free-running conditions. This observation precisely parallels the previously reported effect on behavioral rhythms of the Pdf⁰¹ mutant. PDF is likely to affect some clock neurons directly, since the peptide appears to bind to the surface of many clock neurons, including the LN_vs themselves. We showed that the brain circadian clock in Drosophila is clearly distinguishable from the eyes and other rapidly damping peripheral tissues, as it sustains robust molecular oscillations in DD. At the same time, different clock neurons are likely to work cooperatively within the brain, because the LN_vs alone are insufficient to support the circadian program. Based on the damping results with Pdf⁰¹ mutant flies, we propose that LN_vs, and specifically the PDF neuropeptide that it synthesizes, are important in coordinating a circadian cellular network within the brain. The cooperative function of this network appears to be necessary for maintaining robust molecular oscillations in DD and is the basis of sustained circadian locomotor activity rhythms.     

Male reproduction is affected by RNA interference of period and timeless in the desert locust Schistocerca gregaria

In all living organisms, behavior, metabolism and physiology are under the regulation of a circadian clock. The molecular machinery of this clock has been conserved throughout the animal kingdom. Besides regulating the circadian timing of a variety of processes through a central oscillating mechanism in the brain, these circadian clock genes were found to have a function in peripheral tissues in different insects. Here, we provide evidence that the circadian clock genes period (per) and timeless (tim) have a role in the male locust reproduction. A knockdown of either of the two genes has no effect on male sexual maturation or behavior, but progeny output in their untreated female copulation partners is affected. Indeed, the fertilization rates of the eggs are lower for females with a per or tim RNAi copulation partner as compared to the eggs deposited by females that mated with a control male. As the sperm content of the seminal vesicles is higher in per or tim knockdown males, we suggest that this phenotype could be caused by a disturbance of the circadian regulated sperm transfer in the male reproductive organs, or an insufficient maturation of the sperm after release from the testes.     

Light-break experiments and photoperiodic time measurement in the spider mite Tetranychus urticae

To explain photoperiodic induction of diapause in the spider mite Tetranychus urticae (Acarina: Tetranychidae) a theoretical model was developed, consisting of two components, viz. a “clock” and a photoperiodic “counter” mechanism. The clock executes photoperiodic time measurement according to hourglass kinetics; the counter accumulates the photoperiodic information contained in a number of successive $\text{light}\text{dark}$ cycles by adding up the number of “long” and “short” nights experienced by the developmental stages of the mites sensitive to the photoperiod. The influence of the circadian system on photoperiodic induction is interpreted as an inhibitory effect exerted on the expression of the photoperiodic response; this effect is encountered only in certain photoperiodic regimes, where the circadian system and the photoperiod are out of “resonance” with each other. This “hourglass timer oscillator counter model”, devised to give a theoretical explanation of photoperiodic time measurement, the summation of photoperiodic information, and the influence of the circadian system on photoperiodic induction, proved to be consistent with experimental results obtained with T. urticae in both symmetrical and asymmetrical “skeleton” photoperiods, the latter based on diel as well as non-diel $\text{light}\text{dark}$ cycles.     

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.     

Heterologous Expression and Functional Characterization of a Physcomitrella Pseudo Response Regulator Homolog,PpPRR2, in Arabidopsis

Physcomitrella patens has four homologs of the pseudo-response regulator involved in the circadian clock mechanism in seed plants. To gain insight into their function, Arabidopsis transgenic lines misexpressing PpPRR2 were constructed. Phenotypic analysis of the transformants with reference to clock-related gene expression and photoperiodic responses revealed that heterologous expression of the moss PpPRR2 gene modifies the intrinsic mechanism underlying the circadian clock in Arabidopsis, suggesting that PpPRR2 serves as a clock component in P. patens.     

Immunoreactivities to three circadian clock proteins in two ground crickets suggest interspecific diversity of the circadian clock structure

The closely related crickets Dianemobius nigrofasciatus and Allonemobius allardi exhibit similar circadian rhythms and photoperiodic responses, suggesting that they possess similar circadian and seasonal clocks. To verify this assumption, antisera to Period (PER), Doubletime (DBT), and Cryptochrome (CRY) were used to visualize circadian clock neurons in the cephalic ganglia. Immunoreactivities referred to as PER-ir, DBT-ir, and CRY-ir were distributed mainly in the optic lobes (OL), pars intercerebralis (PI), dorsolateral protocerebrum, and the subesophageal ganglion (SOG). A system of immunoreactive cells in the OL dominates in D. nigrofasciatus, while immunoreactivities in the PI and SOG prevail in A. allardi. Each OL of D. nigrofasciatus contains 3 groups of cells that coexpress PER-ir and DBT-ir and send processes over the frontal medulla face to the inner lamina surface, suggesting functional linkage to the compound eye. Only 2 pairs of PER-ir cells (no DBT-ir) were found in the OL of A. allardi. Several groups of PER-ir cells occur in the brain of both species. The PI also contains DBT-ir and CRY-ir cells, but in A. allardi, most of the DBT-ir is confined to the SOG. Most immunoreactive cells in the PI and in the dorsolateral brain send their fibers to the contralateral corpora cardiaca and corpora allata. The proximity and, in some cases, proven identity of the PER-ir, DBT-ir, and CRY-ir perikarya are consistent with presumed interactions between the examined clock components. The antigens were always found in the cytoplasm, and no diurnal oscillations in their amounts were detected. The photoperiod, which controls embryonic diapause, the rate of larval development, and the wing length of crickets, had no discernible effect on either distribution or the intensity of the immunostaining.     

Role for Circadian Clock Genes in Seasonal Timing: Testing the Bünning Hypothesis

A major question in chronobiology focuses around the “Bünning hypothesis” which implicates the circadian clock in photoperiodic (day-length) measurement and is supported in some systems (e.g. plants) but disputed in others. Here, we used the seasonally-regulated thermotolerance of Drosophila melanogaster to test the role of various clock genes in day-length measurement. In Drosophila, freezing temperatures induce reversible chill coma, a narcosis-like state. We have corroborated previous observations that wild-type flies developing under short photoperiods (winter-like) exhibit significantly shorter chill-coma recovery times (CCRt) than flies that were raised under long (summer-like) photoperiods. Here, we show that arrhythmic mutant strains, per⁰¹, tim⁰¹ and Clk^Jrk, as well as variants that speed up or slow down the circadian period, disrupt the photoperiodic component of CCRt. Our results support an underlying circadian function mediating seasonal daylength measurement and indicate that clock genes are tightly involved in photo- and thermo-periodic measurements.