Yosuke Miyazaki

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.

Thermoperiodic regulation of the circadian eclosion rhythm in the flesh fly, Sarcophaga crassipalpis.

We recorded the eclosion time of the flesh fly, Sarcophaga crassipalpis, at different depths in the outdoor soil and under temperature cycles with various amplitudes in the laboratory, to examine the timing adjustment of eclosion in response to temperature cycles and their amplitudes in the pupal stage. In the soil, most eclosions occurred in the late morning, which was consistent with the eclosion time under pseudo-sinusoidal temperature cycles in the laboratory. The circadian clock controlling eclosion was reset by temperature cycles and free-ran with a period close to 24h. This clock likely helps pupae eclose at an optimal time even when the soil temperature does not show clear daily fluctuations. The eclosion phase of the circadian clock progressively advanced as the amplitude of the pseudo-sinusoidal temperature cycle decreased. This response allows pupae located at any depth in the soil to eclose at the appropriate time despite the depth-dependent phase delay of the temperature change. In contrast, the abrupt temperature increase in square-wave temperature cycles reset the phase of the circadian clock to the increasing time, regardless of the temperature amplitude. The rapid temperature increase may act as the late-morning signal for the eclosion clock.

Circannual rhythm in the varied carpet beetle, Anthrenus verbasci

Although circannual rhythms controlling different physiological processes and various aspects of behavior have been reported in numerous organisms, our understanding of the underlying biological mechanisms is still quite limited. We examined the mechanisms controlling the circannual pupation rhythm of the varied carpet beetle, Anthrenus verbasci. This rhythm is self-sustainable, exhibits temperature compensation of the periodicity, and is entrainable to environmental changes. In addition, the circannual phase response curves to a photoperiod pulse display Type 0 or Type 1 resetting, depending on the duration of the pulse. Thus, we infer that this rhythm is derived from a self-sustaining biological oscillator with a period of about a year, that is, a circannual clock, analogous to the circadian clock. Further, a circadian clock appears to mediate photoperiodic time measurement for phase resetting of the circannual clock. Based on these results and previous research performed in other organisms, we discuss the general characteristics of the physiological mechanisms underpinning circannual rhythmicity.

A circadian system is involved in photoperiodic entrainment of the circannual rhythm of Anthrenus verbasci.

In the circannual pupation rhythm of the varied carpet beetle, Anthrenus verbasci, entrainment to annual cycles is achieved by phase resetting of the circannual oscillator in response to photoperiodic changes. In order to examine whether a circadian system is involved in expression of the periodic pattern and phase resetting of the circannual rhythm as photoperiodic responses, we exposed larvae to light-dark cycles with a short photophase followed by a variable scotophase (the Nanda-Hamner protocol). When the cycle length (T) was a multiple of 24h, i.e., 24, 48, or 72 h, short-day effects were clearer than when T was far from a multiple of 24h, i.e., 36 or 60 h. Exposure to light-dark cycles of T=36 h had effects similar to exposure to long-day cycles of T=24h. The magnitude of phase shifts depended on the duration and the phase of exposure to the cycles of T=36 or 60 h. It was therefore concluded that a circadian system is involved in photoperiodic time measurement for phase resetting of the circannual oscillator of A. verbasci.

Circannual pupation rhythm in the varied carpet beetle Anthrenus verbasci under different nutrient conditions

Anthrenus verbasci pupates in spring and the timing of pupation is controlled by a circannual rhythm. Although A. verbasci is considered to be a univoltine species in Japan, it is assumed that larval development in its natural habitats, including bird nests, varies with nutrient availability, and that the life cycle often takes two or more years to complete. In the present study, larval development and pupation times were compared under constant and outdoor conditions in larvae provided a diet of either high‐nutrient bonito powder or low‐nutrient pigeon feathers. Although a circannual pupation rhythm was observed irrespective of the diet used, larval development was slower on feathers than on bonito powder. The pupation times on feathers varied over three years or more under both constant and outdoor conditions. Under outdoor conditions, larvae grown on feathers needed three years to approach the weight gained within a year by larvae grown on bonito powder. It is considered that life cycle length in A. verbasci is often two years or more in nutritionally unstable natural habitats, and that this species has probably evolved a circannual rhythm as a seasonal adaptation to nutrient‐poor environments.

Temperature cycle amplitude alters the adult eclosion time and expression pattern of the circadian clock gene period in the onion fly

Soil temperature cycles are considered to play an important role in the entrainment of circadian clocks of underground insects. However, because of the low conductivity of soil, temperature cycles are gradually dampened and the phase of the temperature cycle is delayed with increasing soil depth. The onion fly, Delia antiqua, pupates at various soil depths, and its eclosion is timed by a circadian clock. This fly is able to compensate for the depth-dependent phase delay of temperature change by advancing the eclosion time with decreasing amplitude of the temperature cycle. Therefore, pupae can eclose at the appropriate time irrespective of their location at any depth. However, the mechanism that regulates eclosion time in response to temperature amplitude is still unknown. To understand whether this mechanism involves the circadian clock or further downstream physiological processes, we examined the expression patterns of period (per), a circadian clock gene, of D. antiqua under temperature cycles that were square wave cycles of 12-h warm phase (W) and 12-h cool phase (C) with the temperature difference of 8 °C (WC 29:21 °C) and 1 °C (WC 25.5:24.5 °C). The phase of oscillation in per expression was found to commence 3.5h earlier under WC 25.5:24.5 °C as compared to WC 29:21 °C. This difference was in close agreement with the eclosion time difference between the two temperature cycles, suggesting that the mechanism that responds to the temperature amplitude involves the circadian clock.

Exhibition of circannual rhythm under constant light in the varied carpet beetle Anthrenus verbasci

The varied carpet beetle Anthrenus verbasci shows a clear circannual pupation rhythm under light/dark (LD) 12:12. We examined whether this rhythm is exhibited under constant light (LL) of 0.002 Wm−2 and 0.9 Wm−2 intensities. Rhythmic pupation was not observed when the larvae were continuously maintained under LL. Moreover, the circannual rhythmicity of pupation was not observed under LL after pre-exposure to LD 12:12 for 2 or 4 weeks but was observed after exposure for 8 weeks. Under LL of both light intensities, as the pre-exposure to LD 12:12 was longer, the first pupation peaks occurred earlier and almost synchronized with pre-exposure for 8 weeks with the first peak under continuous LD 12:12. However, the transition from LD 12:12 to LL did not reset the phase of this circannual rhythm. Pre-exposure to LD 12:12 probably synchronizes the asynchronous rhythm observed under LL so that a clear circannual pupation rhythm is exhibited.

Circannual Rhythms in Insects

Although many insects adapt to seasonal changes by photoperiodism, a small proportion of insect species use a circannual rhythm for seasonal adaptations. The circannual pupation rhythm of the varied carpet beetle Anthrenus verbasci shows a periodicity of approximately 40 weeks under constant conditions, and the change in photoperiod acts as a zeitgeber. The circannual rhythm of A. verbasci, of which the larval duration varies from one to several years, probably plays an important role for synchronizing the pupation and breeding times with spring each year. There are only slight differences in the critical daylength for circannual entrainment among geographically distinct populations in Japan, and they pupate in the same period under natural conditions in Osaka. Therefore, A. verbasci can adapt to seasonal changes in different regions without changing the parameters of the circannual rhythm. Long-term endogenous rhythms have also been reported in oviposition and pupation of some ant species. These insects are considered to refer not only to external cues but also to the phase of an endogenous clock for maintaining appropriate seasonality.

Circannual pupation timing is not correlated with circadian period in the varied carpet beetle

Anthrenus verbasci (Insecta, Coleoptera, Dermestidae) shows a circannual rhythm in pupation and a circadian rhythm in adult locomotor activity. The period length of the circannual rhythm will be correlated with the length of a circadian period if oscillations of these two rhythms share a common genetic background. We examined the relationship between the two rhythms and found no correlation between the pupation time determined by the circannual rhythm and the circadian period of the adult activity rhythm. This result suggests that the mechanism that produces the circannual rhythm of A. verbasci is not affected by the period of circadian rhythm.

Dependence of phase setting on the amplitude of square-wave and pseudo-sinusoidal temperature cycles in the circadian eclosion rhythm of the onion fly Delia antiqua : Temperature amplitude affects eclosion

With increasing soil depth, the diel temperature amplitude gradually decreases and the phase of the temperature cycle is delayed. The onion fly Delia antiqua (Meigen) (Diptera: Anthomyiidae), which pupates at a soil depth of 2–20 cm, advances the eclosion phase of its circadian rhythm as the temperature amplitude decreases. This ‘temperature amplitude response’ is considered to allow pupae located at any depth to eclose in the early morning. The present study examines the temperature amplitude response of D. antiqua under thermoperiods with amplitudes ranging between 1 and 20 °C. The thermoperiod to which pupae are exposed is either a square‐wave temperature cycle, which is generally used for laboratory experiments, or a pseudo‐sinusoidal temperature cycle, which more closely resembles natural daily temperature cycle, at an average temperature of either 25 or 20 °C. Bursts of eclosion invoked by the abrupt temperature increase in square‐wave cycles are apparently repressed in pseudo‐sinusoidal temperature cycles. Eclosion time shows a clear quantitative shift by 5.9–8.6 h in response to temperature amplitude regardless of the average temperature and the shape of the temperature cycle. The change in the eclosion time is clearly noticeable between amplitudes of 1 and 8 °C but is small between amplitudes of 8 °C or more. Delia antiqua may evolve to compensate for the depth‐dependent phase delay of temperature changes within the possible range of temperature amplitudes in actual soil environments.