Tomoko Ikeno

Photoperiodic diapause under the control of circadian clock genes in an insect

BackgroundMost organisms have evolved a circadian clock in order to anticipate daily environmental changes and many of these organisms are also capable of sophisticated measurement of daylength (photoperiodism) that is used to regulate seasonal events such as diapause, migration and polymorphism. It has been generally accepted that the same elements are involved in both circadian (daily) and seasonal (annual) rhythms because both rely upon daily light-dark cycles. However, as reasonable as this sounds, there remains no conclusive evidence of such a molecular machinery in insects. We have approached this issue by using RNA interference (RNAi) in Riptortus pedestris.ResultsThe cuticle deposition rhythm exhibited the major properties of circadian rhythms, indicating that the rhythm is regulated by a circadian clock. RNAi directed against the circadian clock genes of period and cycle, which are negative and positive regulators in the circadian clock, respectively, disrupted the cuticle deposition rhythm and distinct cuticle layers were produced by these RNAi. Simultaneously, period RNAi caused the insect to avert diapause under a diapause-inducing photoperiod whereas cycle RNAi induced diapause under a diapause-averting photoperiod. The expression patterns of juvenile hormone-regulated genes and the application of juvenile hormone analogue suggested that neither ovarian development itself nor a downstream cascade of juvenile hormone secretion, were disturbed by period and cycle RNAi.ConclusionsThis study revealed that the circadian clock genes are crucial not only for daily rhythms but also for photoperiodic diapause. RNAi directed against period and cycle had opposite effects not only in the circadian cuticle deposition rhythm but also in the photoperiodic diapause. These RNAi also had opposite effects on juvenile hormone-regulated gene expression. It is still possible that the circadian clock genes pleiotropically affect ovarian development but, based on these results, we suggest that the circadian clock operated by the circadian clock genes, period and cycle, governs seasonal timing as well as the daily rhythms.See Commentary: http://www.biomedcentral.com/1741-7007/8/115

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.

Functional circadian clock genes are essential for the overwintering diapause of the Northern house mosquito, Culex pipiens

The short day lengths of late summer are used to program the overwintering adult diapause (dormancy) of the Northern house mosquito, Culex pipiens. Here, we investigated the role of clock genes in initiating this diapause and asked whether the circadian cycling of clock gene expression persists during diapause. We provide evidence that the major circadian clock genes continue to cycle throughout diapause and after diapause has been terminated. RNA interference (RNAi) was used to knock down the core circadian clock genes and to then assess the impact of the various clock genes on the ability of females to enter diapause. RNAi directed against negative circadian regulators (period, timeless and cryptochrome2) caused females that were reared under diapause-inducing, short day conditions to avert diapause. In contrast, knocking down the circadian-associated gene pigment dispersing factor caused females that were reared under diapause-averting, long day conditions to enter a diapause-like state. Our results implicate the circadian clock in the initiation of diapause in C. pipiens.

Molecular characterization of the circadian clock genes in the bean bug, Riptortus pedestris, and their expression patterns under long- and short-day conditions.

Although the molecular mechanisms and the diversity of insect circadian clocks have been well investigated in holometabolous insects, hemimetabolous insects have received little attention. In the present study, we isolated the circadian clock genes, period (per), cycle (cyc), vrille (vri), and mammalian-type cryptochrome (cry-m) from the bean bug Riptortus pedestris. This is the first report of vri and cry-m in hemimetabolous insects. All of the genes showed high similarities to respective homologous genes in other insects. The discovery of cry-m in R. pedestris indicates that the clockwork of hemimetabolous insects is similar to that in insects having CRY-m, including the monarch butterfly Danaus plexippus and the honey bee Apis mellifera, and not to insects lacking it, such as Drosophila melanogaster. Real-time PCR showed that mRNAs of these circadian clock genes exhibited extremely weak diel oscillations at day 9 in the head of R. pedestris, and their expression levels under long- and short-day conditions were nearly identical. In addition, expression levels of per mRNA were almost stable from days 0 to 15 under both photoperiodic conditions. The difference between long-day and short-day conditions in the mRNA level seems too small to distinguish photoperiodic conditions clearly. These results suggest that transcriptional regulations of circadian clock genes would not play an important role in the diapause programming in R. pedestris.

Photoperiodic response requires mammalian-type cryptochrome in the bean bug Riptortus pedestris

The hypothesis that a circadian clock comprised of circadian clock genes is causally involved in insect photoperiodism has been supported by several studies. However, there remains a possibility that the effects of the circadian clock genes on photoperiodism are exerted through pleiotropic (non-circadian) functions provided by each gene independently from its role in the circadian clock. In the present study, we investigated the involvement of the circadian clock gene mammalian-type cryptochrome (cry-m) in photoperiodic regulation of ovarian development in the bean bug Riptortus pedestris by using RNA interference (RNAi). Injection of cry-m double-stranded RNA (dsRNA) induced expression of period (per), whereas did not affect expression of cycle (cyc), showing that CRY-m functions as a negative element on CYC-mediated transcription in the circadian clock. If the circadian clock is indeed involved in photoperiodism, a phenotype produced by RNAi of cry-m will be the same as that produced by RNAi of per, another negative element. The intact insects and insects injected with control dsRNA were found to enter diapause when kept under short-day conditions after adult emergence, while they developed ovaries when kept under long-day conditions after adult emergence. However, cry-m RNAi significantly increased the incidence of reproductive individuals under diapause-inducing short-day conditions, as per RNAi did, in accordance with our expectation.

Causal involvement of mammalian‐type cryptochrome in the circadian cuticle deposition rhythm in the bean bug Riptortus pedestris

Mammalian‐type CRYPTOCHROME (CRY‐m) is considered to be a core repressive component of the circadian clock in various insect species. However, this role is based only on the molecular function of CRY‐m in cultured cells and it therefore remains unknown whether CRY‐m is indispensable for governing physiological rhythms at the organismal level. In the present study, we show that RNA interference (RNAi) targeting of cry‐m in the bean bug Riptortus pedestris disrupts the circadian clock governing the cuticle deposition rhythm and results in the generation of a single cuticle layer. Furthermore, period expression was induced in cry‐m RNAi insects. These results verified that CRY‐m functions as a negative regulator in the circadian clock that generates physiological rhythm at the organismal level.

Circadian clock gene Clock is involved in the photoperiodic response of the bean bug Riptortus pedestris

The role of the circadian clock gene Clock in the circadian rhythm and the photoperiodic regulation of reproductive diapause in the bean bug Riptortus pedestris (Fabricius) (Hemiptera: Alydidae) is investigated. Clock RNA interference (RNAi) disrupts the circadian rhythm in an alternating deposition of polarized and nonpolarized cuticle layers in the endocuticle, and produces only a polarized layer. This indicates that Clock is a core component of the circadian clock and that it acts as a positive element in activating the transcription of downstream genes. In addition, Clock RNAi suppresses ovarian development irrespective of day‐length conditions, which indicates that Clock is involved in the photoperiodic response. The observed phenotypes in Clock RNAi insects, the production of a single polarized layer in the endocuticle and an arrested ovarian development irrespective of photoperiod are the same as those induced by RNAi of cycle, a positive element in the circadian clock. However, the phenotypes induced by RNAi of negative elements period and mammalian‐type cryptochrome differ from those induced by the positive elements. Considering the current data together with previously published findings, it is concluded that the circadian clock, comprising the circadian clock genes, is involved in the photoperiodic response of R. pedestris.

Common features in diverse insect clocks.

This review describes common features among diverse biological clocks in insects, including circadian, circatidal, circalunar/circasemilunar, and circannual clocks. These clocks control various behaviors, physiological functions, and developmental events, enabling adaptation to periodic environmental changes. Circadian clocks also function in time-compensation for celestial navigation and in the measurement of day or night length for photoperiodism. Phase response curves for such clocks reported thus far exhibit close similarities; specifically, the circannual clock in Anthrenus verbasci shows striking similarity to circadian clocks in its phase response. It is suggested that diverse biological clocks share physiological properties in their phase responses irrespective of period length. Molecular and physiological mechanisms are best understood for the optic-lobe and mid-brain circadian clocks, although there is no direct evidence that these clocks are involved in rhythmic phenomena other than circadian rhythms in daily events. Circadian clocks have also been localized in peripheral tissues, and research on their role in various rhythmic phenomena has been started. Although clock genes have been identified as controllers of circadian rhythms in daily events, some of these genes have also been shown to be involved in photoperiodism and possibly in time-compensated celestial navigation. In contrast, there is no experimental evidence indicating that any known clock gene is involved in biological clocks other than circadian clocks.

Involvement of the brain region containing pigment-dispersing factor-immunoreactive neurons in the photoperiodic response of the bean bug, Riptortus pedestris

The concept of insect photoperiodism based on a circadian clock has been supported by many studies demonstrating that the behavioural circadian rhythm and the photoperiodic response are driven by the same circadian clock genes. However, the neuronal mechanism of the circadian clock underlying photoperiodism is poorly understood. To examine whether circadian rhythm and photoperiodism share a neuronal mechanism, we focused on the neurons that express neuropeptide pigment-dispersing factor (PDF) in the bean bug, Riptortus pedestris. PDF has been identified as an important regulator of the insect circadian rhythm and is expressed in circadian clock neurons of various insect species. In R. pedestris, PDF immunoreactivity was detected in some clusters of cells and their fibres in the optic lobe and the protocerebrum. cDNA encoding a PDF precursor protein was highly conserved between R. pedestris and many other insects. Differences between day and night were not observed in the immunolabelling intensity in cell bodies of PDF-immunoreactive neurons and pdf mRNA expression levels in the head. Surgical removal of the region containing PDF-immunoreactive cell bodies at the medulla disrupted the photoperiodic regulation of diapause. However, gene suppression of pdf by RNA interference did not affect the photoperiodic response. These results suggest that the region containing PDF-immunoreactive somata is important for the photoperiodic response in R. pedestris, but pdf mRNA expression is probably not required for the response.

Continuous activity and no cycling of clock genes in the Antarctic midge during the polar summer.

The extreme seasonal shifts of day length in polar regions, ranging from constant light in the summer to constant darkness in the winter, pose an intriguing environment for probing activity rhythms and the functioning of circadian clocks. Here, we monitor locomotor activity during the summer on the Antarctic Peninsula and under laboratory conditions, as well as the accompanying patterns of clock gene expression in the Antarctic midge, the only insect endemic to Antarctica. Larvae and adults are most active during the warmest portion of the day, but at a constant temperature they remain continuously active regardless of the photoregime, and activity also persists in constant darkness. The canonical clock genes period, timeless, Clock, and vrille are expressed in the head but we detected no cycling of expression in either the field or under diverse photoregimes in the laboratory. The timekeeping function of the clock has possibly been lost, enabling the midge to opportunistically exploit the unpredictable availability of permissive thermal conditions for growth, development, and reproduction during the short summer in Antarctica.