The molecular mechanisms of circadian rhythms are well known, but how multiple clocks within one organism generate a structured rhythmic output remains a mystery. Many animals show bimodal activity rhythms with morning (M) and evening (E) activity bouts. One long-standing model assumes that two mutually coupled oscillators underlie these bouts and show different sensitivities to light. Three groups of lateral neurons (LN) and three groups of dorsal neurons govern behavioral rhythmicity of Drosophila. Recent data suggest that two groups of the LN (the ventral subset of the small LN cells and the dorsal subset of LN cells) are plausible candidates for the M and E oscillator, respectively. We provide evidence that these neuronal groups respond differently to light and can be completely desynchronized from one another by constant light, leading to two activity components that free-run with different periods. As expected, a long-period component started from the E activity bout. However, a short-period component originated not exclusively from the morning peak but more prominently from the evening peak. This reveals an interesting deviation from the original Pittendrigh and Daan (1976) model and suggests that a subgroup of the ventral subset of the small LN acts as "main" oscillator controlling M and E activity bouts in Drosophila.

The fruit fly, Drosophila melanogaster, shows a clear circadian locomotor rhythm in light cycles and constant darkness. Although the rhythm disappears in constant light, we found that temperature cycles drive the circadian rhythm both in locomotor activity and molecular abundance of PERIOD (PER) and TIMELESS (TIM). The thermoperiodically induced locomotor rhythm entailed an anticipatory activity at the late thermophase, which required several transient cycles to establish a steady-state entrainment, suggesting that the rhythm is endogenous and driven by a circadian clock. Western blot analysis revealed that PER and TIM increased during the cryophase, peaking at the middle to late cryophase. PER was also cyclically expressed under the temperature cycle in the known per-expressing neurons, i.e. so-called lateral (LNs) and dorsal neurons (DNs), and two pairs of cells (LPNs) that were located in the lateral posterior protocerebrum. It is thus suggested that the temperature cycle induces the cycling of PER and TIM either by blocking somewhere in the photic entrainment pathway during the cryophase or temporally activating their translation to sufficient protein levels to drive a circadian oscillation. In flies lacking pigment-dispersing factor (PDF) or PDF-expressing cells, the anticipatory activity was relatively dispersed. disco(2) mutant flies lacking the lateral neurons still showed an anticipatory activity, but with dispersed activity. These behavioural results suggest that not only LNs but also DNs and LPNs can, at least, partially participate in regulating the thermoperiodically induced rhythm.

The fruit fly Drosophila melanogaster shows a bimodal circadian locomotor rhythm with peaks at lights-on and before lights-off, which are regulated by multiple clocks in the brain. Even under light-dark cycles, the timing of the evening peak is highly dependent on temperature, starting earlier under lower ambient temperature but terminating almost at the same time. In the present study, using behavioral and immunohistochemical assays, the authors show that separate groups of clock neurons, either light-entrainable or temperature-entrainable, form a functional system driving the locomotor rhythm. When subjected to a light cycle combined with a temperature cycle advanced by 6 h relative to the light cycle, the dorsally located neurons (DNs) and lateral posterior neurons (LPNs) shifted their phase of TIMELESS expression, but the laterally located protocerebral neurons (LNs) basically maintained their original phase. Thus, the LNs seem to be preferentially light-entrainable and the DNs and LPNs to be primarily temperature-entrainable. In pdf(01) mutant flies that lack the neuropeptide PDF in the ventral groups of LNs, the onset of the evening peak was greatly advanced even under synchronized light and temperature cycles and was shifted even more than in wild-type flies in response to a 6-h phase shift of the temperature cycle, suggesting that ventral LNs have a strong impact on the phase of the other cells. It seems likely that the 2 sets of clock cells with different entrainability to light and temperature, and the coupling between them, enable Drosophila to keep a proper phase relationship of circadian activity with respect to the daily light and temperature cycles.

Recent studies revealed that the neuronal network controlling overt rhythms shows striking similarity in various insect orders. The pigment-dispersing factor seems commonly involved in regulating locomotor activity. However, there are considerable variations in the molecular oscillatory mechanism, and input and output pathways among insects. In Drosophila, autoregulatory negative feedback loops that consist of clock genes, such as period and timeless are believed to create 24-h rhythmicity. Although similar clock genes have been found in some insects, the behavior of their product proteins shows considerable differences from that of Drosophila. In other insects, mammalian-type cryptochrome (cry2) seems to work as a transcriptional repressor in the feedback loop. For photic entrainment, Drosophila type cryptochrome (cry1) plays the major role in Drosophila while the compound eyes are the major photoreceptor in others. Further comparative study will be necessary to understand how this variety of clock mechanisms derived from an ancestral one.

Periodic expression of so-called clock genes is an essential part of the circadian clock. In Drosophila melanogaster the cyclic expression of per and tim through an autoregulatory feedback loop is believed to play a central role in circadian rhythm generation. However, it is still elusive whether this hypothesis is applicable to other insect species. Here it is shown that per gene plays a key role in the rhythm generation in the cricket Gryllus bimaculatus. Measurement of per mRNA levels in the optic lobe revealed the rhythmic expression of per in light cycles with a peak in the late day to early night, persisting in constant darkness. A single injection of per double-stranded RNA (dsRNA) into the abdomen of the final instar nymphs effectively knocked down the mRNA levels as adult to about 50% of control animals. Most of the per dsRNA-injected crickets completely lost the circadian locomotor activity rhythm in constant darkness up to 50 days after the injection, whereas those injected with DsRed2 dsRNA as a negative control clearly maintained it. The electrical activity of optic lobe efferents also became arrhythmic in the per dsRNA-injected crickets. These results not only suggest that per plays an important role in the circadian rhythm generation also in the cricket but also show that RNA interference is a powerful tool to dissect the molecular machinery of the cricket circadian clock.

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Although butterflies undergo a dramatic morphological transformation from larva to adult via a pupal stage (holometamorphosis), crickets undergo a metamorphosis from nymph to adult without formation of a pupa (hemimetamorphosis). Despite these differences, both processes are regulated by common mechanisms that involve 20-hydroxyecdysone (20E) and juvenile hormone (JH). JH regulates many aspects of insect physiology, such as development, reproduction, diapause, and metamorphosis. Consequently, strict regulation of JH levels is crucial throughout an insect’s life cycle. However, it remains unclear how JH synthesis is regulated. Here, we report that in the corpora allata of the cricket, Gryllus bimaculatus, Myoglianin (Gb’Myo), a homolog of Drosophila Myoglianin/vertebrate GDF8/11, is involved in the down-regulation of JH production by suppressing the expression of a gene encoding JH acid O-methyltransferase, Gb’jhamt. In contrast, JH production is up-regulated by Decapentaplegic (Gb’Dpp) and Glass-bottom boat/60A (Gb’Gbb) signaling that occurs as part of the transcriptional activation of Gb’jhamt. Gb’Myo defines the nature of each developmental transition by regulating JH titer and the interactions between JH and 20E. When Gb’myo expression is suppressed, the activation of Gb’jhamt expression and secretion of 20E induce molting, thereby leading to the next instar before the last nymphal instar. Conversely, high Gb’myo expression induces metamorphosis during the last nymphal instar through the cessation of JH synthesis. Gb’myo also regulates final insect size. Because Myo/GDF8/11 and Dpp/bone morphogenetic protein (BMP)2/4-Gbb/BMP5–8 are conserved in both invertebrates and vertebrates, the present findings provide common regulatory mechanisms for endocrine control of animal development.

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