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Keywords:

  • cuticle deposition rhythm;
  • diapause;
  • photoperiodic counter;
  • photoperiodic response;
  • photoperiodic time measurement;
  • Riptortus pedestris

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Photoperiodic Time Measurement and Circadian Clock
  5. Molecular Machinery of the Circadian Clock Governing Circadian Rhythmicity
  6. Mutants or Variants Lacking Circadian Rhythmicity or Photoperiodism
  7. Effects of Silencing of Clock Genes on Photoperiodism in a Fly and a Cricket
  8. Modular Pleiotropy and Gene Pleiotropy
  9. Causal Involvement of Circadian Clock Genes in Photoperiodism in the Bean Bug
  10. Contrary Evidence Regarding the Involvement of Circadian Clock Genes in Photoperiodism
  11. Reading the Clock
  12. Conclusion and Future Directions
  13. Acknowledgments
  14. References

Functional involvement of a circadian clock in photoperiodism for measuring the length of day or night had been proposed more than 70 years ago, and various physiological experiments have supported the idea. However, the molecular basis of a circadian clock has remained veiled in insects. Nevertheless, our knowledge of the functional elements of a circadian clock governing circadian rhythmicity has advanced rapidly. Since both circadian rhythms and photoperiodism depend on the daily cycles of environmental changes, it is easy to assume that the same clock elements are involved in both processes. Recently, the RNA interference (RNAi) technique clarified that the molecular machinery of a circadian clock governing photoperiodism is identical to that governing circadian rhythmicity. Here, I review the theoretical background of photoperiodic responses incorporating a circadian clock(s) and recent progress on the molecular clockwork involved in photoperiodism in the bean bug Riptortus pedestris and other insect species. I have focused on the intense controversy regarding the involvement of a circadian clock in insect photoperiodism.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Photoperiodic Time Measurement and Circadian Clock
  5. Molecular Machinery of the Circadian Clock Governing Circadian Rhythmicity
  6. Mutants or Variants Lacking Circadian Rhythmicity or Photoperiodism
  7. Effects of Silencing of Clock Genes on Photoperiodism in a Fly and a Cricket
  8. Modular Pleiotropy and Gene Pleiotropy
  9. Causal Involvement of Circadian Clock Genes in Photoperiodism in the Bean Bug
  10. Contrary Evidence Regarding the Involvement of Circadian Clock Genes in Photoperiodism
  11. Reading the Clock
  12. Conclusion and Future Directions
  13. Acknowledgments
  14. References

There are several predominant environmental rhythms on the earth: the daily day–night cycle caused by the earth's rotation about its own axis and the annual seasonal cycle caused by the earth's revolution around the sun. Temporal coordination with both these rhythms is crucial for the maintenance of fitness in organisms. Thus, most organisms have evolved a circadian clock, which is set by light to time various daily activities at the biochemical, physiological and behavioral levels; further, some organisms, especially those inhabiting the temperate zone, have evolved photoperiodism, which is a physiological reaction to the length of day or night for the timing of development, reproduction and diapause in anticipation of seasonal changes in the environment (Tauber et al. 1986; Danks 1987). More than 70 years ago, Bünning (1936) first proposed the functional involvement of a circadian clock in the photoperiodic clock for measuring the length of day or night (Bünning's hypothesis). Sophisticated experimental designs, including night interruption as well as Bünsow and Nanda–Hamner protocols, have also verified the involvement of a circadian clock in photoperiodism in various organisms (Beck 1980; Pittendrigh 1981; Saunders 2002; Putterill et al. 2010). Thus, the involvement of a circadian clock in photoperiodism is generally accepted in various organisms. In addition, in plants, fungi and mammals, it is considered that a series of circadian clock genes regulating circadian rhythmicity governs photoperiodism (reviewed by Hazlerigg 2010; Putterill et al. 2010; Ronnenberg et al. 2010). However, in insects, there is no conclusive evidence on this issue; in fact, there is an intense controversy regarding Bünning's hypothesis (see Danks 2005; Bradshaw & Holzapfel 2007a, b).

Recently, the RNA interference (RNAi) technique clarified that the molecular machinery of a circadian clock governing photoperiodism is identical to that governing circadian rhythmicity. In this work, I have reviewed the theoretical background of photoperiodic responses incorporating a circadian clock(s) and recent progress on the molecular clockwork involved in photoperiodism in the bean bug Riptortus pedestris (Fabricius) and some other insect species. I have focused on the intense controversy regarding the involvement of a circadian clock in insect photoperiodism.

Photoperiodic Time Measurement and Circadian Clock

  1. Top of page
  2. Abstract
  3. Introduction
  4. Photoperiodic Time Measurement and Circadian Clock
  5. Molecular Machinery of the Circadian Clock Governing Circadian Rhythmicity
  6. Mutants or Variants Lacking Circadian Rhythmicity or Photoperiodism
  7. Effects of Silencing of Clock Genes on Photoperiodism in a Fly and a Cricket
  8. Modular Pleiotropy and Gene Pleiotropy
  9. Causal Involvement of Circadian Clock Genes in Photoperiodism in the Bean Bug
  10. Contrary Evidence Regarding the Involvement of Circadian Clock Genes in Photoperiodism
  11. Reading the Clock
  12. Conclusion and Future Directions
  13. Acknowledgments
  14. References

A photoperiodic response in insects comprises a sequence of several events: (i) photoreception; (ii) measurement of day or night length by a photoperiodic time measurement system; (iii) simultaneous counting of the number of photoperiodic conditions by a counter system; and (iv) action of the endocrine effectors that determine seasonal events like diapause. Bünning (1936) first proposed the idea that circadian rhythms regulating daily activities form the basis of the photoperiodic time measurement system. He proposed that the 24 h of a day are composed of two 12-h half-cycles: the photophil (or the light-requiring phase) and the scotophil (or the dark-requiring phase). Short-day effects are seen when light is restricted to the photophil, while long-day effects are produced when light penetrates the scotophil. Although his idea is too simple to explain the range of photoperiodic responses in various organisms, the basic concept of the idea, i.e., the functional involvement of a circadian clock in photoperiodic time measurement, is now widely accepted, not only in insects (Saunders & Bertossa 2011) but also in various organisms from fungi to mammals (Hazlerigg 2010; Putterill et al. 2010; Ronnenberg et al. 2010).

Experiments designed to reveal the possible role of the circadian system in photoperiodic time measurement are based on the known effects of environmental light pulses on the phase shifting and entrainment of circadian oscillations. The experiments include Nanda–Hamner and Bünsow experiments and others (for full details, see Saunders 2002, 2010). In Nanda–Hamner experiments (resonance experiments) (Nanda & Hamner 1958), a “short-day” light phase (e.g. 10 or 12 h) is coupled with varying lengths of darkness (e.g. 4–72 h). These abnormal light–dark cycles are then repeated throughout the photoperiod-sensitive period of insects, and the results are expressed as the proportion of each sample entering diapause. In insects showing “positive” circadian resonance, recurrent peaks and troughs of high and low diapause incidence, respectively, are observed (Saunders 2002). In Bünsow experiments (Bünsow 1953), insects are exposed to 48- or 72-h cycles containing a “short” main photophase (e.g. 12 h) and a supplementary light pulse of short duration, systematically interrupting, in separate experimental subsets, a greatly extended “night.” These abnormal light cycles are repeated throughout the photoperiod-sensitive period, and diapause incidence is assessed in each experimental group at the end of the experiment. In both Nanda–Hamner and Bünsow experiments, circadian involvement in the photoperiodic reaction is suggested when alternating peaks and troughs of diapause incidence are observed at an interval of approximately 24 h in the greatly extended night; the lack of such periodicity is usually taken as evidence for a non-circadian or hourglass-like timer.

Nearly a dozen photoperiodic clock models have been established by incorporating accumulated experimental data under various photoperiodic conditions (Vaz Nunes & Saunders 1999). They are mainly divided into two categories: hourglass timer and the circadian clock model. One of the well-known examples of an hourglass timer is the photoperiodic response of the aphid Megoura viciae Buckton (Lees 1973). In the extensive studies by Lees, no evidence for circadian rhythmicity was obtained. However, Saunders (2009) clearly pointed out that hourglass-like responses are the expression of a heavily dampened circadian oscillator. Some additional experiments revealed the role of a circadian oscillator in photoperiodic timing, even in M. viciae (Vaz Nunes & Hardie 1993).

Circadian clock models have shown several variations. In flesh flies, Sarcophaga spp., an influential explanation is that time measurement is based on a circadian oscillator, which sets its phase at dusk and positions the definitive light-sensitive phase (ϕi) at the latter half of the scotophase. During summer, ϕi is exposed to light, and therefore, insects recognize the conditions as long days and produce a non-diapause phenotype. On the other hand, during autumn, ϕi is not exposed to light, and therefore, insects recognize the conditions as short days and enter diapause (Saunders 1979; see also Goto & Numata 2009; Tagaya et al. 2010). This model is called “the external coincidence model” and was first described by Pittendrigh and Minis (1964).

“The internal coincidence model” (Pittendrigh 1960; Tyshchenko 1966) is most applicable to a photoperiodic response in the parasitic wasp Nasonia vitripennis (Walker) (Saunders 1974). This model proposes two oscillators entrained by dawn and dusk, respectively, whose internal phase relationship changes with the length of the photophase. Induction of a long-day response occurs according to the “overlap” between the particular phases of the two components (see Danilevsky et al. 1970). The females of N. vitripennis were maintained in light–dark cycles ranging in length from 12 to 72 h, with a 4- to 28-h photophase. This resonance experiment revealed the periodic maxima of diapause induction, at an interval of about 24 h. Interestingly, the “ascending slopes” of these maxima appeared to obtain their principal time cue from dusk and the “descending slopes” from dawn (Saunders 1974). These results suggest that two independent oscillators, dawn and dusk, are involved in the Nasonia photoperiodic clock.

The external and internal coincidence models are applicable to qualitative time measurement, and thus they simply distinguish between “long” and “short” nights, relative to a critical length. However, some models have incorporated a quantitative concept (Zaslavski 1988, 1996; Vaz Nunes 1998). Observations with various insects have indicated that long nights and short nights are more fundamentally different than simply “long” and “short,” respectively. To reflect such phenomena, Vaz Nunes (1998) proposed “the double circadian oscillator model,” in which there are two circadian oscillations, each determining the length of a night and each assigning it a quantitative value. The model is able to explain various photoperiodic responses under natural and unnatural photoperiodic conditions.

As evident, about a dozen photoperiodic clock models have been established, and most have assumed the causal involvement of a circadian system in photoperiodic time measurement. The hourglass timer can also be explained by a heavily dampened circadian oscillator. Thus, causal involvement of a circadian clock in photoperiodic time measurement is now widely accepted, although some discrepancies still exist.

Molecular Machinery of the Circadian Clock Governing Circadian Rhythmicity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Photoperiodic Time Measurement and Circadian Clock
  5. Molecular Machinery of the Circadian Clock Governing Circadian Rhythmicity
  6. Mutants or Variants Lacking Circadian Rhythmicity or Photoperiodism
  7. Effects of Silencing of Clock Genes on Photoperiodism in a Fly and a Cricket
  8. Modular Pleiotropy and Gene Pleiotropy
  9. Causal Involvement of Circadian Clock Genes in Photoperiodism in the Bean Bug
  10. Contrary Evidence Regarding the Involvement of Circadian Clock Genes in Photoperiodism
  11. Reading the Clock
  12. Conclusion and Future Directions
  13. Acknowledgments
  14. References

Although the molecular machinery of the circadian clock involved in photoperiodism has long been veiled, our knowledge of the functional elements of the circadian clock governing circadian rhythmicity has advanced rapidly.

One of the landmark studies in chronobiology is that by Konopka and Benzer (1971), who documented three mutants in Drosophila melanogaster Meigen with abnormal circadian rhythms of adult eclosion and locomotor activity. Interestingly, the mutations were all mapped to the same gene, known as period (per). This discovery further led to the discovery of at least a half-dozen other such “circadian clock genes.”

Interlocked positive and negative feedback loops based on transcription and translation are the essence of circadian clocks in insects (Fig. 1; Tomioka et al. 2012). In Drosophila, the core interlocked feedback loop is established by the CLOCK (CLK) and CYCLE (CYC) proteins, per and timeless (tim) mRNAs, and the PER and TIM proteins. CYC and CLK act as positive regulators and form a heterodimer to induce the transcription of per, tim and other output genes, whereas PER and TIM act as negative regulators and form a heterodimer to suppress CYC–CLK activity. The feedback loop produces oscillation in the expression levels of some clock components. The per and tim mRNA levels are low during photophase but high during scotophase. The PER and TIM levels also show similar patterns, but their peaks are delayed by a few hours as compared with those of the corresponding mRNA levels. CRYPTOCHROME (CRY) plays a pivotal role in circadian clocks in animals. Two types of CRY are known in insects: Drosophila-type CRY (CRY-d) and mammalian-type CRY (CRY-m) (Sandrelli et al. 2008). Drosophila does not possess cry-m in its genome, but non-drosophilid insects examined thus far possess cry-m. CRY-d is a flavin-based UV- and blue-light-sensitive photopigment and causes degradation of TIM in a light-dependent manner. Thus, CRY-d is not a core component of the circadian clock, at least in the central clock of D. melanogaster. On the other hand, CRY-m does not function as a photoreceptor, but it acts as a transcriptional repressor. From a series of experiments on the monarch butterfly Danaus plexippus (L.), Reppert and his colleagues (Yuan et al. 2007; Zhu et al. 2008) established a circadian clock model for insects by incorporating CRY-m. In this model, TIM and PER bind to CRY-m to stabilize CRY-m, and the complex represses CYC–CLK-mediated transcription. Thus, CRY-m functions as a core component of the circadian clock and acts as a negative regulator, like PER and TIM.

figure

Figure 1. Circadian clock models for Drosophila melanogaster and the monarch butterfly Danaus plexippus. In Drosophila, CLK and CYC act as positive regulators to induce not only per and tim but also other circadian clock genes responsible for other interlocked feedback loops of the clock (clockwork orange [cwo], Par-domain protein 1 [Pdp1] and vrille [vri]) and clock-controlled genes (ccg) responsible for downstream cascades. On the other hand, PER and TIM act as negative regulators to inhibit transcriptional activity of CYC–CLK. CRY-d causes degradation of TIM in a light-dependent manner. The clockwork in D. melanogaster and D. plexippus are nearly identical, except that in D. plexippus, CRY-m, which is not found in the genome of Drosophila, forms a complex with PER and TIM to inhibit the transcriptional activity of CYC–CLK. Thus, CRY-m also functions as a negative regulator in this model. Modified from Yuan et al. (2007), Zhu et al. (2008), and Tomioka et al. (2012).

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Mutants or Variants Lacking Circadian Rhythmicity or Photoperiodism

  1. Top of page
  2. Abstract
  3. Introduction
  4. Photoperiodic Time Measurement and Circadian Clock
  5. Molecular Machinery of the Circadian Clock Governing Circadian Rhythmicity
  6. Mutants or Variants Lacking Circadian Rhythmicity or Photoperiodism
  7. Effects of Silencing of Clock Genes on Photoperiodism in a Fly and a Cricket
  8. Modular Pleiotropy and Gene Pleiotropy
  9. Causal Involvement of Circadian Clock Genes in Photoperiodism in the Bean Bug
  10. Contrary Evidence Regarding the Involvement of Circadian Clock Genes in Photoperiodism
  11. Reading the Clock
  12. Conclusion and Future Directions
  13. Acknowledgments
  14. References

Riihimaa and Kimura (1988) isolated a strain lacking photoperiodic diapause, from natural populations of Chymomyza costata (Zetterstedt). The non-photoperiodic-diapause (npd) mutant does not respond to photoperiods and continues development irrespective of photoperiods. It is noteworthy that circadian gating of adult eclosion is also lost in npd mutants (Lankinen & Riihimaa 1992; Koštál & Shimada 2001). Genetic linkage analysis clearly demonstrated that the gene responsible for the non-diapause phenotype mapped to the locus including tim (Pavelka et al. 2003). The tim mRNA is barely detectable in npd-mutant flies (Pavelka et al. 2003; Stehlík et al. 2008). In npd mutants, per mRNA oscillation is greatly suppressed as compared with that in the wild-type flies (Koštál & Shimada 2001).

Goto et al. (2006) documented a variant strain displaying arrhythmic adult eclosion in the flesh fly Sarcophaga bullata (Parker): the variant ecloses arrhythmically throughout the photophase and scotophase, rather than eclosing in early photophase, as observed in the wild-type flies. Concomitantly, the variant flies fail to respond to short days to enter pupal diapause. The loss of both diapause (a photoperiodic response) and gating of adult eclosion (a circadian rhythm) suggests that the same clock system is involved in both the responses. An examination of the expression patterns of per and tim has demonstrated that the levels of both per and tim mRNAs are higher in the variant than in the wild type.

The linden bug Pyrrhocoris apterus (L.) also shows a clear photoperiodic response of reproductive diapause. Hodková et al. (2003) focused on circadian behavior, clock gene expression and diapause induction in a strain that failed to enter diapause even under diapause-inducing short-day conditions. Interestingly, the diurnal activity peak under short-day conditions in this strain is similar to that under long-day conditions in the photoperiod-sensitive strain. The wild-type strain expressed ten-fold higher levels of per mRNA under short-day conditions than under long-day conditions. Interestingly, the variant exhibited low levels of per mRNA irrespective of the photoperiodic conditions, and the levels were comparable with those observed in the wild-type strain under long-day conditions. These results indicate that malfunction of a circadian clock evokes long-day-type responses, not only the circadian response but also the photoperiodic response, irrespective of photoperiods.

Yamada and Yamamoto (2011) focused on the genetic association of the circadian and photoperiodic clocks in Drosophila triauraria Bock & Wheeler. They analyzed the association of five circadian clock genes (per, tim, Clk, cyc and cry-d) with the occurrence of diapause by crossing a strain showing a clear photoperiodic response with a non-diapause strain found in low latitudes. Single-nucleotide polymorphism and deletion analyses of the five circadian clock genes in the backcross progeny revealed that allelic differences in tim and cry-d between the strains were additively associated with the differences in the incidence of diapause.

These results suggest that the circadian clock genes governing circadian rhythmicity are somehow involved in photoperiodism. However, we detected merely correlation, and it remains uncertain whether the relationships between circadian rhythmicity and photoperiodic responses are causal. Recently, several authors approached this issue by using RNAi. RNAi is a powerful tool for analyzing the functions of individual genes, especially in non-model organisms, in which it has almost been impossible to analyze the functions of genes (Mito et al. 2011).

Effects of Silencing of Clock Genes on Photoperiodism in a Fly and a Cricket

  1. Top of page
  2. Abstract
  3. Introduction
  4. Photoperiodic Time Measurement and Circadian Clock
  5. Molecular Machinery of the Circadian Clock Governing Circadian Rhythmicity
  6. Mutants or Variants Lacking Circadian Rhythmicity or Photoperiodism
  7. Effects of Silencing of Clock Genes on Photoperiodism in a Fly and a Cricket
  8. Modular Pleiotropy and Gene Pleiotropy
  9. Causal Involvement of Circadian Clock Genes in Photoperiodism in the Bean Bug
  10. Contrary Evidence Regarding the Involvement of Circadian Clock Genes in Photoperiodism
  11. Reading the Clock
  12. Conclusion and Future Directions
  13. Acknowledgments
  14. References

Disruption of photoperiodism with RNAi of circadian clock genes has been documented in the larval photoperiodic diapause of the drosophilid fly C. costata (Pavelka et al. 2003) and in the photoperiodic control of nymphal development in the cricket Modicogryllus siamensis Chopard (Sakamoto et al. 2009). In C. costata, tim RNAi produced a non-diapause phenotype even under diapause-inducing short-day conditions, although its effect was very small (Pavelka et al. 2003). In M. siamensis, per RNAi caused arrhythmic locomotor activity under constant darkness as well as light–dark conditions. Interestingly, irrespective of photoperiod, the adult emergence patterns in per RNAi insects were not similar to those in either intact or control insects kept under long days or short days but to those in insects kept under constant darkness (Sakamoto et al. 2009; Fig. 2). Thus, RNAi of circadian clock genes affected photoperiodic responses in a fly and a cricket, two systematically distant insect species.

figure

Figure 2. Adult emergence patterns in Modicogryllus siamensis hatched from eggs laid by a female injected with double-stranded RNA (dsRNA) of per (dsper) or Red2 of Discosoma sp. (dsRed2; negative control) or from eggs laid by an intact female, under conditions of 16 hours light : 8 hours dark (16L:8D), 8L:16D or constant darkness (DD). Based on Sakamoto et al. (2009).

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Modular Pleiotropy and Gene Pleiotropy

  1. Top of page
  2. Abstract
  3. Introduction
  4. Photoperiodic Time Measurement and Circadian Clock
  5. Molecular Machinery of the Circadian Clock Governing Circadian Rhythmicity
  6. Mutants or Variants Lacking Circadian Rhythmicity or Photoperiodism
  7. Effects of Silencing of Clock Genes on Photoperiodism in a Fly and a Cricket
  8. Modular Pleiotropy and Gene Pleiotropy
  9. Causal Involvement of Circadian Clock Genes in Photoperiodism in the Bean Bug
  10. Contrary Evidence Regarding the Involvement of Circadian Clock Genes in Photoperiodism
  11. Reading the Clock
  12. Conclusion and Future Directions
  13. Acknowledgments
  14. References

Modular pleiotropy is defined as the effect of a module on phenotype, whereas gene pleiotropy is defined as the effect of an individual gene on phenotype (Fig. 3). According to the concept of modular pleiotropy, malfunction of circadian clock genes affects photoperiodism by altering the clock function. Alternatively, according to the concept of gene pleiotropy, malfunction of circadian clock genes directly affects diapause, and the effect is not mediated by a circadian clock but by individual genes (Emerson et al. 2009). It is quite difficult to distinguish the two concepts in the results of RNAi experiments for analyzing effects on photoperiodism, at least at this stage, because both concepts can be applied to the abovementioned examples (C. costata and M. siamensis). Therefore, we can not exclude the possibility that clock genes themselves directly affected diapause in these organisms (gene pleiotropy), and not through a malfunction of the circadian clock. In R. pedestris, however, the results strongly support the concept that a circadian clock operated by circadian clock genes governs photoperiodism. In the following section, I review recent progress on the role of clock genes in photoperiodism in R. pedestris by using RNAi.

figure

Figure 3. Modular pleiotropy and gene pleiotropy. Each circle indicates a single circadian clock gene and the circadian clocks established by the genes are shown as hexagrams. The closed circle indicates a gene of interest. (A) Modular pleiotropy. Mutation in a circadian clock gene (closed circle) or silencing of its function modifies the function of the circadian clock (large circle with broken line), and this modified function of the circadian clock produces a different photoperiodic response. Thus, modular pleiotropy occurs when a gene(s) exerts multiple phenotypic effects only indirectly by modifying the functional expression of the module in which it plays a role. (B) Gene pleiotropy. Gene pleiotropy occurs when a single gene affects more than one phenotype. In this case, mutation in a circadian clock gene (closed circle) or silencing of its function may or may not affect the clock function, but the gene(s) (encircled by broken line) directly affect(s) photoperiodism. Based on Emerson et al. (2009).

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Causal Involvement of Circadian Clock Genes in Photoperiodism in the Bean Bug

  1. Top of page
  2. Abstract
  3. Introduction
  4. Photoperiodic Time Measurement and Circadian Clock
  5. Molecular Machinery of the Circadian Clock Governing Circadian Rhythmicity
  6. Mutants or Variants Lacking Circadian Rhythmicity or Photoperiodism
  7. Effects of Silencing of Clock Genes on Photoperiodism in a Fly and a Cricket
  8. Modular Pleiotropy and Gene Pleiotropy
  9. Causal Involvement of Circadian Clock Genes in Photoperiodism in the Bean Bug
  10. Contrary Evidence Regarding the Involvement of Circadian Clock Genes in Photoperiodism
  11. Reading the Clock
  12. Conclusion and Future Directions
  13. Acknowledgments
  14. References

Photoperiodism and circadian rhythm in the bean bug

The bean bug R. pedestris, formerly known as Riptortus clavatus (Thunberg) (see Kikuhara 2005), is a major pest of grain legumes in Africa and Asia (Panizzi et al. 2000) (Fig. 4A). It exhibits a clear photoperiodic response for the induction of adult reproductive diapause. Females develop ovaries and begin to lay eggs promptly after adult emergence under long-day conditions, whereas ovarian development is suppressed under short-day conditions (Numata & Hidaka 1982; Fig. 4B). Photoperiodic sensitivity persists even after adult emergence in this species (Numata 1990). In diapause females, transcription of two yolk protein genes, cyanoprotein-α (CP-α) and vitellogenin (Vg), is suppressed (Hirai et al. 1998; Miura et al. 1998). In males, a secretory fluid accumulates in the ectodermal accessory gland reservoir and mating behavior is induced under long-day conditions, whereas under short-day conditions, the reservoir is deflated and mating behavior is suppressed (Numata & Kobayashi 1989; Fig. 4C–E). In both sexes, adult diapause is primarily due to the cessation of juvenile hormone (JH) secretion by the corpus allatum (CA) (Numata & Hidaka 1984; Morita & Numata 1997; Morita 1999).

figure

Figure 4. Riptortus pedestris and its internal reproductive organs. (A) Adult female. (B) Ovaries. Under long-day conditions, light-blue yolk is deposited in the oocytes, and mature eggs are ovulated into the oviduct (left). Under short-day conditions, no deposition of light-blue yolk is observed in the oocytes (right). of, oocyte; od, oviduct; e, egg. (C) Testes and accessory glands of a male. The reservoir is filled with secretory fluid under long-day conditions (D) and is deflated under short-day conditions (E). t, testis; e.g., ectodermal accessory gland; r, reservoir. Scale bar, 500 μm. (A,B) are kindly provided by Dr H Numata of Kyoto University and Dr T Ikeno of Ohio State University, respectively. (C–E) are from Ikeno et al. (2011c).

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Although this species shows diel activity patterns in feeding, locomotor activity, mating and oviposition under light–dark cycles, a free-running rhythm is shown only in oviposition (Kadosawa 1982, 1983; Numata & Matsui 1988). However, the oviposition rhythm is easily disturbed by manipulation and is not observed in diapause individuals. Therefore, the oviposition rhythm is not a good indicator of circadian rhythmicity in this species.

In some insect species, a cuticle deposition rhythm is controlled by a circadian clock, and one pair of cuticle layers is deposited every day to generate the cuticle (Neville 1975; Ito et al. 2008). The cuticle of the insect exoskeleton is composed of the epicuticle, the exocuticle and the endocuticle. The endocuticle thickens by alternating the deposition of two types of layers with different orientations of chitin microfibrils: (i) lamellate layers, in which the microfibrils are secreted helicoidally from the epidermal cells; and (ii) non-lamellate layers, in which they are secreted unidirectionally. In R. pedestris, the cuticle deposition rhythm free-runs under constant conditions (self-sustaining oscillation), the number of deposited cuticle layers varies with the given number of temperature cycles (entrainment to environmental cycles) and the Q10 value of the rhythm is close to 1.0 (temperature compensation). Thus, the rhythm exhibits the major properties of a circadian rhythm, indicating that the cuticle deposition rhythm in R. pedestris is regulated by a circadian clock (Ikeno et al. 2010).

RNAi of circadian clock genes in the bean bug

Ikeno et al. (2008) identified several genes from R. pedestris, which are highly similar to the circadian clock genes in other insect species. My colleagues and I performed RNAi by injecting double-stranded RNA (dsRNA) into the head or abdomen of the female adults of R. pedestris. Injection of per, cyc and cry-m, dsRNA disrupted the circadian clock regulating the cuticle deposition rhythm and produced only one type of cuticle layer. The results revealed that per, cyc and cry-m are core components of the circadian clock in R. pedestris. Interestingly, per and cry-m RNAi and cyc RNAi produced distinct types of cuticle layers: the former produced a thick non-lamellate layer, whereas the latter produced a thick lamellate layer (Fig. 5; Ikeno et al. 2010, 2011a). The distinct phenotypes produced by per and cry-m RNAi and cyc RNAi are reasonable when we consider the roles of these genes in the circadian clock model (Fig. 1): per and cry-m function as negative regulators that suppress the CYC–CLK activity, whereas cyc acts as a positive regulator that induces the expression of downstream cascade genes as well as per. The per and cry-m RNAi fail to suppress CYC–CLK activity, causing activation of clock-controlled genes regulating the downstream cascade, whereas cyc RNAi directly suppresses CYC–CLK activity, thus suppressing the downstream cascade. Thus, both per and cry-m RNAi and cyc RNAi arrest the clock, but at distinct phases. In R. pedestris, cry-m RNAi produces high levels of per mRNA, while cyc RNAi produces low levels of per mRNA. These results also support our conclusion (Ikeno et al. 2011a,b).

figure

Figure 5. The endocuticle of the hind leg of Riptortus pedestris. (A) Cross-sections of the tibia of the hind leg of intact, per RNAi, and cyc RNAi insects at 20 days after adult emergence under short-day conditions. Alternating double layers (lamellate and non-lamellate layers) are clearly observed in intact individuals. Arrows indicate lamellate layers. Double-stranded RNA (dsRNA) was injected on the day of adult emergence. per RNAi produced a single thickened non-lamellate layer and cyc RNAi produced a single thickened lamellate layer. Scale bar, 25 μm. (B) Cuticle layers were observed at 20 days after the injection of per, cyc or β-lactamase (bla; negative control) dsRNA or saline. The ordinate shows the percentage of individuals with alternating cuticle layers in the endocuticle. Bars with the same letters indicate no significant difference (Tukey-type multiple comparisons for proportions, P > 0.05). The cry-m RNAi also produced the same phenotype as per RNAi (data not shown; see Ikeno et al. 2011a). Based on Ikeno et al. (2010).

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Photoperiodic response was also disrupted by RNAi of the circadian clock genes in R. pedestris females: per and cry-m RNAi induced ovarian development even under diapause-inducing short-day conditions, whereas cyc RNAi suppressed ovarian development even under diapause-averting long-day conditions (Ikeno et al. 2010, 2011b; Fig. 6). These different phenotypes induced by per and cry-m RNAi and cyc RNAi indicate that arrest of the circadian clock at distinct phases by RNAi activates distinct cascades involved in the photoperiodic response, as observed in the case of the cuticle deposition rhythm.

figure

Figure 6. Effects of per and cyc RNAi on ovarian development in Riptortus pedestris. The experimental schedules are shown as horizontal hatched bars (short-day conditions) and open bars (long-day conditions). Arrowheads indicate the days of adult emergence and arrows indicate the days of dissection. Double-stranded RNA (dsRNA) was injected on the day of adult emergence. Insects were maintained continuously under short-day conditions (A), transferred from long-day to short-day conditions (B), maintained continuously under long-day conditions (C) or transferred from short-day to long-day conditions (D). Bars with the same letters indicate no significant difference (Tukey-type multiple comparisons for proportions, P >0.05). The result of cry-m RNAi was nearly identical to that of per RNAi (data not shown; see Ikeno et al. 2011b). For further explanation, see Figure 5. Based on Ikeno et al. (2010).

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My colleagues and I also performed Clk RNAi. Clk is considered to be a positive regulator in the clock model (Fig. 1). In females of R. pedestris, Clk RNAi produced the same phenotype as cyc RNAi, not only for the cuticle deposition rhythm but also for the photoperiodic response (T Ikeno, K Ishikawa, H Numata, SG Goto, unpubl. data, 2012). These results also support our conclusion.

The next focus is the endocrine effector. Although adult diapause in R. pedestris is due to a cessation of JH secretion (Numata & Hidaka 1984; Morita & Numata 1997; Morita 1999), it is still not possible to measure the JH concentration in this species. One of the reasons is that JH synthetic activity in the CA of R. pedestris becomes very low in vitro and is almost undetectable. In addition, the CA of hemipteran insects, including this species, synthesizes a novel JH (Kotaki 1993; Kotaki et al. 2009), and a system to quantify JH concentrations in the hemolymph remains to be established. As an alternative approach, it is possible to estimate the JH concentration by examining expression of JH-regulated genes; in R. pedestris, the expression of CP-α and Vg transcripts are induced by JH, whereas transferrin (Tf) expression is suppressed by JH (Hirai et al. 1998, 2000; Miura et al. 1998). CP-α and Vg transcripts were detectable in females injected with saline and reared under long-day conditions, whereas they were barely detected in cyc RNAi insects. By contrast, Tf transcript was detected in diapause females injected with cyc dsRNA, whereas it was undetectable in control females reared under long-day conditions. In addition, a JH analog application induced ovarian development in cyc RNAi females. Thus, circadian clock genes are neither involved in the process directly regulating ovarian development nor in the cascade of events downstream from JH secretion, but they are involved in an upstream event.

Further, the effects of per and cyc RNAi on the photoperiodic response and cuticle deposition rhythm were examined in males of R. pedestris. The cuticle deposition rhythm was also disrupted in males injected with per and cyc dsRNA, and per and cyc RNAi produced a thick non-lamellate layer and a lamellate layer, respectively, as in females. The per RNAi induced the accumulation of the secretory fluid even under short-day conditions, whereas cyc RNAi suppressed its accumulation even under long-day conditions (Ikeno et al. 2011c). This indicates that a circadian clock is involved in a common mechanism governing photoperiodic responses in both sexes.

Thus, RNAi experiments in R. pedestris clearly revealed that a circadian clock operated by circadian clock genes, namely, per, cry-m, cyc and Clk, governs seasonal timing as well as daily rhythms.

Contrary Evidence Regarding the Involvement of Circadian Clock Genes in Photoperiodism

  1. Top of page
  2. Abstract
  3. Introduction
  4. Photoperiodic Time Measurement and Circadian Clock
  5. Molecular Machinery of the Circadian Clock Governing Circadian Rhythmicity
  6. Mutants or Variants Lacking Circadian Rhythmicity or Photoperiodism
  7. Effects of Silencing of Clock Genes on Photoperiodism in a Fly and a Cricket
  8. Modular Pleiotropy and Gene Pleiotropy
  9. Causal Involvement of Circadian Clock Genes in Photoperiodism in the Bean Bug
  10. Contrary Evidence Regarding the Involvement of Circadian Clock Genes in Photoperiodism
  11. Reading the Clock
  12. Conclusion and Future Directions
  13. Acknowledgments
  14. References

In contrast to the recent progress in RNAi experiments, there is some contrary evidence regarding the involvement of circadian clock genes in photoperiodism.

Progress in Drosophila melanogaster

Saunders et al. (1989) first studied the role of circadian clock genes in photoperiodism by using mutants of Drosophila melanogaster. They showed that null mutants of per and deletion strains of per loci were able to discriminate photoperiods for the induction of diapause, irrespective of the complete absence of their locomotor activity rhythms under constant conditions (Fig. 7). This clearly indicates that per is not necessary for the photoperiodic induction of diapause in this species. There are, however, two alternative explanations. The central circadian pacemaker remains at least residually functional in null mutants of per and can thus support photoperiodic sensitivity. This explanation is supported by the presence of a per-independent oscillator in D. melanogaster, as proposed in previous studies (e.g. Helfrich-Förster et al. 2001). Another explanation is that clock genes (or a single gene) other than per are involved, which continue their photoperiodic functions even when the functionality of the circadian pacemaker is severely compromised. This explanation was explicitly favored by Saunders et al. (1989) (for details, see Koštál 2011).

figure

Figure 7. Photoperiodic response curves in wild-type (Canton-S, solid line) and per01 mutant (broken line) of Drosophila melanogaster at 12°C. The mutant showing arrhythmic locomotor activity was capable of discriminating photoperiods. The critical daylength of the mutant seems to be shifted to be approximately 2 h shorter than that of the wild type, but Emerson et al. (2009) reanalyzed the data and found no significant differences in the critical photoperiods between the lines. Based on Saunders et al. (1989).

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Tauber et al. (2007) clearly demonstrated that allelic variation in tim is responsible for the diapause capability of D. melanogaster. Natural populations of D. melanogaster in Europe and North Africa show a latitudinal cline in the frequencies of two major alleles of tim, ls-tim and s-tim. The former produces both the long L-TIM and the short S-TIM, whereas the latter produces only the short S-TIM. Interestingly, introduction of one of the natural or artificial alleles of tim into different genetic backgrounds slightly but clearly affected the incidence of diapause, with females having ls-tim showing higher diapause incidence than those having s-tim. In addition, there was no significant effect of an interaction between photoperiod and the different tim alleles on the incidence of diapause. This demonstrated that photoperiod and tim exert their influence on diapause independently. Furthermore, a null mutant of tim, tim01, is also capable of entering diapause. These results indicate that tim is not causally essential for expression of diapause but its allele can increase or decrease diapause incidence. Sandrelli et al. (2007) further investigated characteristics of the s-tim and ls-tim alleles and clarified that the ability of LS-TIM to interact with CRY-d is much weaker than that of S-TIM to interact with CRY-d, resulting in flies having ls-tim showing significantly smaller phase responses in locomotor activity than those having s-tim. Thus, in D. melanogaster, tim plays two roles: (i) a central role in the circadian clock; and (ii) a regulation of diapause incidence. On the basis of these results, Bradshaw and Holzapfel (2007b) stated that the quest for understanding the photoperiodic timer by conducting exhaustive studies on specific circadian clock genes has shown little promise. However, it is noteworthy that although tim01 flies showed an ability to enter diapause, they failed to show a photoperiodic response. The flies enter diapause at a certain proportion irrespective of the photoperiodic conditions (Tauber et al. 2007; Fig. 8). Therefore, the results could be interpreted to suggest that tim is still essential for photoperiodic time measurement to discriminate short- and long-day conditions.

figure

Figure 8. Photoperiodic response of Drosophila melanogaster. (A) Photoperiodic response of cantonized wild-type (open circles) and tim01 (closed circles) flies. The tim01 flies lost the ability of producing a significant response to photoperiod. (B) Photoperiodic response of hemizygous ls-tim transformant (open circles) and w;tim01 (closed circles). A significant difference was observed between the genotypes. Based on Tauber et al. (2007).

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Progress in Drosophila littoralis and Wyeomyia smithii

Lankinen and Forsman (2006) investigated genetic linkage between the circadian clock and photoperiodic time measurement system in Drosophila littoralis (Meigen). A northern strain shows a long critical daylength for diapause, an early phase of the entrained eclosion rhythm under extremely short days, and a short period for the free-running eclosion rhythm. A southern strain shows a short critical daylength, a late eclosion phase, and a long free-running period. These distinct strains were crossed to determine the linkage between photoperiodic and circadian characteristics. After 54 generations, which involved free recombination, artificial selection and genetic drift, a novel strain having “southern” diapause and “northern” eclosion rhythm characteristics was produced. The complete separation of eclosion rhythm characteristics from photoperiodism reveals that the systems controlling the variability of the eclosion rhythm and photoperiodism are different and genetically separable in D. littoralis.

In the pitcher-plant mosquito Wyeomyia smithii (Coquilett), the critical daylength for larval diapause increases regularly with latitude and altitude (Bradshaw & Holzapfel 2001a,b). However, neither the period nor the amplitude of the circadian rhythm response to the Nanda–Hamner protocol is correlated with the critical photoperiod (Bradshaw et al. 2003, 2006). In addition, by conducting crossing experiments, Mathias et al. (2006) revealed that genetic modification of a circadian clock does not form the basis for adaptive modification of photoperiodic time measurement among geographic strains. Moreover, quantitative trait loci (QTL) analyses revealed that tim is not causally involved in differences in the critical photoperiod, but rather only epistatically interacts with the critical photoperiod (Mathias et al. 2007). Recently, Bradshaw et al. (2012) investigated the genetic linkage between the critical daylength and rhythmic response to the Nanda–Hamner protocol. In W. smithii, there exists a negative correlation between the two parameters. By antagonistic selection against this genetic correlation, the correlation rapidly breaks down.

If the same suite of circadian clock genes that is responsible for expression of seasonal timing is also responsible for expression of daily cycles, then adaptive modification of photoperiodic time measurement necessarily implies genetic modification of the circadian clock. Under this assumption, the results obtained for D. littoralis and W. smithii indicate that circadian rhythmicity does not form the causal basis for the adaptive divergence of photoperiodic time measurement. However, another explanation is still possible. It is obvious that downstream cascades governing circadian behaviors and photoperiodic responses are completely different. The free-running period is directly linked to characteristics of a circadian clock, and therefore selections made during the course of artificial and natural selection are obvious. However, for other characteristics, we know the phenotypes that were selected, but we do not know the mechanisms that were selected. Thus, it is possible to cause adaptive divergence of photoperiodic time measurement without changing the physiological characteristics of a circadian clock, by reading the hands of the clock. This is implied by Lankinen and Forsman (2006) themselves. In the next section, I discuss a few models producing different critical photoperiods with no alteration of the circadian oscillator.

Reading the Clock

  1. Top of page
  2. Abstract
  3. Introduction
  4. Photoperiodic Time Measurement and Circadian Clock
  5. Molecular Machinery of the Circadian Clock Governing Circadian Rhythmicity
  6. Mutants or Variants Lacking Circadian Rhythmicity or Photoperiodism
  7. Effects of Silencing of Clock Genes on Photoperiodism in a Fly and a Cricket
  8. Modular Pleiotropy and Gene Pleiotropy
  9. Causal Involvement of Circadian Clock Genes in Photoperiodism in the Bean Bug
  10. Contrary Evidence Regarding the Involvement of Circadian Clock Genes in Photoperiodism
  11. Reading the Clock
  12. Conclusion and Future Directions
  13. Acknowledgments
  14. References

One model (Fig. 9) is based on a qualitative evaluation of photoperiod under external coincidence (Bradshaw et al. 1998; Tagaya et al. 2010). The model can explain the difference in critical daylength without assuming alteration of the physiological characteristics of the circadian oscillator. The model assumes that ϕi is located at the late scotophase in strain X, but in another strain, strain Y, it is located slightly earlier (Fig. 9A). Under short-day conditions (Fig. 9A-1), both strains enter diapause because ϕi is in darkness, whereas under long-day conditions (Fig. 9A-3), they avert diapause because ϕi is exposed to light. Under an intermediate photoperiod (Fig. 99-2), ϕi is delayed sufficiently to coincide with the dawn transition of the main photophase in strain X, but not in strain Y. Thus, the model incorporating no change in the oscillator itself is able to explain the difference in critical daylength (Fig. 9B).

figure

Figure 9. Conceptual diagram of the difference in the qualitative evaluation of photoperiods between strains having different critical daylengths (Tagaya et al. 2010). (A) The hypothesis is based on the external coincidence model. In this example, two strains, X and Y, possess an identical circadian oscillator, but the positions of their photoinducible phase, ϕi, are different: ϕi of strain Y locates slightly earlier than that of strain X. Under short-day conditions (A-1), both the strains enter diapause because ϕi is in darkness, whereas under long-day conditions (A-3), they avert diapause because ϕi is exposed to light. Under an intermediate photoperiod (A-2), ϕi is delayed sufficiently to coincide with the dawn transition of the main photophase in strain X, but not in strain Y. Thus, the critical daylength of strain Y is longer than that of strain X (B).

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Tagaya et al. (2010) further proposed other models (Figs 10,11). The models are based on the quantitative evaluation of photoperiodic time measurement. One assumes alteration of the internal threshold for the hypothetical substance that induces diapause (Fig. 10), and the other assumes alteration of the accumulation rates of the hypothetical substance (Fig. 11). Neither assumes alteration of the physiological characteristics of the circadian oscillator involved in photoperiodic time measurement, but they can explain inter-strain differences in critical daylength. During the photoperiod-sensitive stage, insects accumulate or degrade the hypothetical substance to induce diapause. The substance is similar to the “diapause titer” proposed by Gibbs (1975) or “INDSUM” proposed by Lewis and Saunders (1987). The substance is accumulated under short-day conditions but is degraded under long-day conditions. When the level of the accumulated substance exceeds a certain internal threshold, diapause induction is determined, whereas non-diapause development is determined when the accumulation is below the threshold (Gibbs 1975). If we focus on the calendar day rather than substance accumulation, the threshold can be translated into the concept of “the required day number” (RDN). RDN is defined as the number of calendar days required to raise the proportion of short-day or long-day responses (Saunders 1971).

figure

Figure 10. Conceptual diagrams of a model under the assumption of difference in the quantitative evaluation of photoperiods. This model incorporates a difference in the internal threshold between two strains, that is, the threshold is higher in strain X than in strain Y, but it does not assume alteration of the physiological characteristics of the circadian oscillator. (A) The hypothetical substance is synthesized or degraded according to photoperiod in a quantitative manner. (B) According to the photoperiod applied, the hypothetical substance is accumulated in a quantitative manner. Diapause is determined when the amount exceeds the threshold, while non-diapause is determined, when the amount does not exceed the threshold. Under short days (a in A), the hypothetical substance is synthesized at a high rate, the insects accumulate enough amount of the substance at the end of the photoperiod-sensitive stage (a in B), and thus, both the strains enter diapause (a in C). Under long days (c in A), however, the hypothetical substance is degraded at a high rate, the accumulation is below the threshold of both the strains (c in B), and thus, both the strains fail to enter diapause (c in C). On the other hand, under an intermediate photoperiod (b in A), the accumulation exceeds the threshold of strain Y but not that of strain X (b in B), and thus, strain Y enters diapause, but strain X does not (b in C). Thus, there arises a difference in the critical daylength between the strains.

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figure

Figure 11. Conceptual diagrams of a model under the assumption of difference in the quantitative evaluation of photoperiods. This model incorporates inter-strain difference in the synthesis and degradation rates of the diapause titer (the hypothetical substance). (A) Strain X shows a lower synthesis rate and a higher degradation rate of the hypothetical substance than strain Y under short and long days, respectively. (B) According to the photoperiod applied, the hypothetical substance is accumulated or degraded in a quantitative manner. Because of the higher synthesis rate, a higher amount of the substance is accumulated in strain Y than in strain X, and thus, strain Y tends to enter diapause even under the same photoperiod. Thus, there arises a difference in the critical daylength between the strains (C). For further explanation, see Figure 10.

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In the model shown in Figure 10, the internal threshold of strain X is higher than that of strain Y. Under short days (a in Fig. 10A), the hypothetical substance is synthesized at a high rate, the insects accumulate a sufficient amount of the substance at the end of the photoperiod-sensitive stage (a in Fig. 10B), and thus, both the strains enter diapause (a in Fig. 10C). Under long days (c in Fig. 10A), however, the hypothetical substance is degraded at a high rate, the accumulation is below the threshold of both strains (c in Fig. 10B), and thus, both strains fail to enter diapause (c in Fig. 10C). On the other hand, under an intermediate photoperiod (b in Fig. 10A), the accumulation exceeds the threshold of strain Y but not that of strain X (b in Fig. 10B), and thus, strain Y enters diapause, but strain X does not (b in Fig. 10C). Alternatively, alteration of the accumulation or degradation rates of the hypothetical substance also explains the difference in critical daylength. In the model shown in Figure 11, strain X shows a lower synthesis rate and a higher degradation rate of the hypothetical substance than strain Y under short and long days, respectively. According to the photoperiod applied, the hypothetical substance is accumulated or degraded in a quantitative manner. Strain Y accumulates a higher amount of the substance than strain X, and thus, it tends to enter diapause even under the same photoperiod. Thus, there arises a difference in the critical daylength between the strains.

The important point is that segregation of characteristics related to photoperiodism and circadian rhythmicity is not necessary to deny a causal linkage between a circadian clock and photoperiodic time measurement. If organisms are able to read a clock and respond correctly according to the hands of the clock, they do not need to change the wheel gears of the clock. Otherwise, they do not favor a change in the clock itself because the phase relationship of the clock with the environmental cycles is still important for other phenotypes. Even if a single core oscillator or several oscillators sharing the same physiological mechanisms are responsible for both photoperiodic responses and circadian behaviors, the oscillator can possibly produce different responses through distinct downstream pathways.

Conclusion and Future Directions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Photoperiodic Time Measurement and Circadian Clock
  5. Molecular Machinery of the Circadian Clock Governing Circadian Rhythmicity
  6. Mutants or Variants Lacking Circadian Rhythmicity or Photoperiodism
  7. Effects of Silencing of Clock Genes on Photoperiodism in a Fly and a Cricket
  8. Modular Pleiotropy and Gene Pleiotropy
  9. Causal Involvement of Circadian Clock Genes in Photoperiodism in the Bean Bug
  10. Contrary Evidence Regarding the Involvement of Circadian Clock Genes in Photoperiodism
  11. Reading the Clock
  12. Conclusion and Future Directions
  13. Acknowledgments
  14. References

The present review emphasizes that circadian clock genes, which are responsible for a circadian clock governing circadian rhythmicity, also govern photoperiodism, not by their pleiotropic effects but as a clock. However, thus far, we have only a few examples supporting this concept, and therefore, we do not know whether the concept is generally applicable. Further comparative studies are obviously needed. It is also important to note that the hypothesis emphasized here does not imply that the oscillators governing circadian rhythmicity and photoperiodism are identical. Distinct oscillators sharing identical components may govern circadian rhythmicity and photoperiodism. Indeed, in D. melanogaster and other insects, circadian clock genes are expressed in various neurons in the brain (Helfrich-Förster 1995; Shao et al. 2008; Yang et al. 2009; Muguruma et al. 2010).

In addition, we have to pay considerable attention to insect diversity (Saunders 2011, 2012). Data from various species clearly demonstrate that several types of photoperiodic mechanisms exist in insects. Although both the external and internal coincidence models assume involvement of a circadian clock in photoperiodism, the models indicate greatly different modes of action, i.e., the former hypothesizes a single core oscillator and two roles for light, whereas the latter hypothesizes two oscillators and a single role for light. In some species, photoperiodic time measurement is accomplished by a circadian clock, which heavily dampens out during a long night. Moreover, we have to bear in mind the diversity of molecular clockwork among insects. For example, tim and cry-d are not found in the genomes of the honeybee Apis mellifera L and N. vitripennis, whereas cry-m is not found in the genome of D. melanogaster. In contrast to the pivotal role of tim in Drosophila, its role seems less important in the locomotor activity rhythm of the cricket Gryllus bimaculatus De Geer (Danbara et al. 2010), even though oscillation of its expression is as great as that of per expression. However, tim is important in the firebrat Thermobia domestica (Packard), early branched insects within the insect lineage (Kamae & Tomioka 2012). Thus, the roles of circadian clock genes in a circadian clock and the roles of a circadian clock itself in photoperiodism may differ among insects.

After verifying the roles of a circadian clock in photoperiodism, we next address the question regarding the location of the clock and the way of its involvement in photoperiodism. The site of the circadian clock involved in photoperiodism has not been well characterized. RNAi experiments or experiments with circadian clock gene mutants or variants themselves have not been able to provide the relevant information. Many studies have shown the importance of the brain with regard to photoperiodism, and thus the clock is considered to be located in the brain neurons. In the blow fly Protophormia terraenovae (Robineau–Desvoidy), surgical ablation of a small area of the brain and immunocytochemical analysis revealed that small ventral lateral neurons (s-LNvs), which are PER and pigment-dispersing factor (PDF) immunoreactive, govern both circadian behavior and photoperiodic induction of diapause (Shiga & Numata 2009). However, such neuroanatomical studies themselves can not provide information on the molecular machinery in the neurons. RNAi experiments combined with neuroanatomy will offer a very important piece of information on the physiological mechanisms of photoperiodism.

The pathways linking a circadian clock with the photoperiodic response cascade remain to be clarified in insects, whereas they have been elucidated in plants and mammals (see Dardente et al. 2010; Hut 2010; Imaizumi 2010; Masumoto et al. 2010). A reverse genetic approach with circadian clock genes provides valuable information, but its potential is limited. An approach involving circadian clock genes is the only window to gain an insight into photoperiodic response. In addition to reverse genetic approaches, forward genetic approaches are needed. Identifying “good” mutants by large-scale mutagenesis, like the approach adopted in circadian clock analysis by Konopka and Benzer (1971), as well as creating loss-of-function transformants by using transposon-mediated technologies, deciphering the genetic background of geographic variants showing different characteristics with regard to photoperiodism, and RNA sequencing of short day- and long day-exposed individuals with high-throughput sequencing technologies would greatly contribute to understanding the whole cascade of physiological mechanisms underpinning photoperiodism.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Photoperiodic Time Measurement and Circadian Clock
  5. Molecular Machinery of the Circadian Clock Governing Circadian Rhythmicity
  6. Mutants or Variants Lacking Circadian Rhythmicity or Photoperiodism
  7. Effects of Silencing of Clock Genes on Photoperiodism in a Fly and a Cricket
  8. Modular Pleiotropy and Gene Pleiotropy
  9. Causal Involvement of Circadian Clock Genes in Photoperiodism in the Bean Bug
  10. Contrary Evidence Regarding the Involvement of Circadian Clock Genes in Photoperiodism
  11. Reading the Clock
  12. Conclusion and Future Directions
  13. Acknowledgments
  14. References

I thank T Ikeno of Ohio State University, H Numata of Kyoto University, Y Miyazaki of Ashiya University, MT Kimura of Hokkaido University and S Shiga of Osaka City University for their invaluable suggestions and critical reading of the manuscript. This work was supported by a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Photoperiodic Time Measurement and Circadian Clock
  5. Molecular Machinery of the Circadian Clock Governing Circadian Rhythmicity
  6. Mutants or Variants Lacking Circadian Rhythmicity or Photoperiodism
  7. Effects of Silencing of Clock Genes on Photoperiodism in a Fly and a Cricket
  8. Modular Pleiotropy and Gene Pleiotropy
  9. Causal Involvement of Circadian Clock Genes in Photoperiodism in the Bean Bug
  10. Contrary Evidence Regarding the Involvement of Circadian Clock Genes in Photoperiodism
  11. Reading the Clock
  12. Conclusion and Future Directions
  13. Acknowledgments
  14. References
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