The circadian clock generates rhythms of approximately 24 h through periodic expression of the clock genes. In insects, the major clock genes period (per) and timeless (tim) are rhythmically expressed upon their transactivation by CLOCK/CYCLE, with peak levels in the early night. In Drosophila, clockwork orange (cwo) is known to inhibit the transcription of per and tim during the daytime to enhance the amplitude of the rhythm, but its function in other insects is largely unknown. In this study, we investigated the role of cwo in the clock mechanism of the cricket Gryllus bimaculatus. The results of quantitative RT-PCR showed that under a light/dark (LD) cycle, cwo is rhythmically expressed in the optic lobe (lamina-medulla complex) and peaks during the night. When cwo was knocked down via RNA interference (RNAi), some crickets lost their locomotor rhythm, while others maintained a rhythm but exhibited a longer free-running period under constant darkness (DD). In cwoRNAi crickets, all clock genes except for cryptochrome 2 (cry2) showed arrhythmic expression under DD; under LD, some of the clock genes showed higher mRNA levels, and tim showed rhythmic expression with a delayed phase. Based on these results, we propose that cwo plays an important role in the cricket circadian clock.

Insects living in the temperate zone enter a physiological state of arrested or slowed development to overcome an adverse season, such as winter. Developmental arrest, called diapause, occurs at a species-specific developmental stage, and embryonic and pupal diapauses have been extensively studied in mostly holometabolous insects. Some other insects overwinter in the nymphal stage with slow growth for which the mechanism is poorly understood. Here, we show that this nymphal period of slow growth is regulated by temperature and photoperiod through separate pathways in the cricket Modicogryllus siamensis. The former regulates the growth rate, at least in part, through the insulin / target of rapamycin (TOR) signaling pathway. Lower temperature down-regulates the expression of insulin-like peptide (Ms’Ilp) and Target of rapamycin (Ms’Tor) genes to slow down the growth rate without affecting the number of molts. The latter regulates the number of molts independent of temperature. Short days increase the number of molts through activation of the juvenile hormone (JH) pathway and down-regulation of myoglianin (Ms’myo), a member of the TGFβ family, which induces adult metamorphosis. In contrast, long days regulate Ms’myo expression to increase during the fifth to sixth instar to initiate adult metamorphosis. When Ms’myo expression is suppressed, juvenile hormone O-methyl transferase (Ms’jhamt) was up-regulated and increased molts to prolong the nymphal period even under long-day conditions. The present findings suggested that the photoperiod regulated Ms’myo, and the JH signaling pathway and the temperature-controlled insulin/TOR pathway cooperated to regulate nymphal development for overwintering to achieve seasonal adaptation of the life cycle in M. siamensis.

Diapause is a strategy used by many insects living in temperate zones to survive the winter. The most reliable environmental cue to induce diapause is photoperiod, for which day or night length is measured by a mechanism called the photoperiodic clock. Despite several studies in insects, the photoperiodic mechanisms remain unclear. In the present study, we show that cryptochromes (crys) mediate photoperiodic responses in the cricket Modicogryllus siamensis. Nymphal development of M. siamensis is highly dependent on photoperiod: under long‐day conditions, most nymphs become adults within 60 days after hatching by undergoing seven moults, whereas, under short‐day conditions, they take much longer (approximately 180 days), with an increased number of moults. Similar to most insects, the cricket has two cry genes: the Drosophila type cry (Ms'cry1) and the mammalian type cry (Ms'cry2). Ms'cry1 shows some constitutive expression, whereas Ms'cry2 is rhythmically expressed under both long‐day and short‐day conditions. Parental RNA interference against either or both of the two cry genes partially prevents the long‐day responses, whereas inhibition of Ms'cry1 or Ms'cry1/Ms'cry2 enhances the short‐day response with a further delay of adult emergence or its complete prevention, respectively. Nymphal RNA interference of Ms'crys at the fourth‐instar stage delays adult emergence and increases the number of moults under long‐day conditions. We hypothesize that Ms'cry genes are involved not only in the photoperiodic mechanism, but also in the regulatory mechanism of nymphal development downstream of the photoperiodic clock in the cricket M. siamensis.

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