Fecundity is optimized by levels of nutrient signal-dependent expression of Dve and EcR in Drosophila male accessory gland.

Steroid hormones play various physiological roles including metabolism and reproduction. Steroid hormones in insects are ecdysteroids, and the major form in Drosophila melanogaster is ecdysone. In Drosophila males, the accessory gland is responsive to nutrient-dependent regulation of fertility/fecundity. The accessory gland is composed of two types of binucleated epithelial cells: a main cell and a secondary cell (SC). The transcription factors Defective proventriculus (Dve), Abdominal-B, and Ecdysone receptors (EcRs) are strongly expressed in adult SCs. We show that this EcR expression is regulated by parallel pathways of nutrient signaling and the Dve activity. Induction of Dve expression is also dependent on nutrient signaling, and it becomes nutrient signal-independent during a restricted period of development. Forced dve expression during the restricted period significantly increased the number of SCs. Here, we provide evidence that the level of nutrient signal-dependent Dve expression during the restricted period determines the number of SCs, and that ecdysone signaling is also crucial to optimize male fecundity through nutrient signal-dependent survival and maturation of SCs.

Reciprocal Coupling of Circadian Clocks in the Compound Eye and Optic Lobe in the Cricket Gryllus bimaculatus.

The circadian system comprises multiple clocks, including central and peripheral clocks. The central clock generally governs peripheral clocks to synchronize circadian rhythms throughout the animal body. However, whether the peripheral clock influences the central clock is unclear. This issue can be addressed through a system comprising a peripheral clock (compound eye clock [CE clock]) and central clock (the optic lobe [OL] clock) in the cricket Gryllus bimaculatus. We previously found that the compound eye regulates the free-running period (τ) and the stability of locomotor rhythms driven by the OL clock, as measured by the daily deviation of τ at 30°C. However, the role of the CE clock in this regulation remains unexplored. In this study, we investigated the importance of the CE clock in this regulation using RNA interference (RNAi) of the period (per) gene localized to the compound eye (perCE-RNAi). The perCE-RNAi abolished the compound eye rhythms of the electroretinogram (ERG) amplitude and clock gene expression but the locomotor rhythm driven by the OL clock was maintained. The locomotor rhythm of the tested crickets showed a significantly longer τ and greater daily variation of τ than those of control crickets treated with dsDsRed2. The variation of τ was comparable with that of crickets with the optic nerve severed. The τ was considerably longer but was comparable with that of crickets with the optic nerve severed. These results suggest that the CE clock regulates the OL clock to maintain and stabilize τ.

Fbxl4 Regulates the Photic Entrainment of Circadian Locomotor Rhythms in the Cricket Gryllus bimaculatus.

Photic entrainment is an essential property of the circadian clock that sets the appropriate timing of daily behavioral and physiological events. However, the molecular mechanisms underlying the entrainment remain largely unknown. In the cricket Gryllus bimaculatus, the immediate early gene c-fosB plays an important role in photic entrainment, followed by a mechanism involving cryptochromes (crys). However, the association between c-fosB expression and crys remains unclear. In the present study, using RNA-sequencing analysis, we found that five Fbxl family genes (Fbxl4, Fbxl5, Fbxl16, Fbxl-like1, and Fbxl-like2) encoding F-box and leucine-rich repeat proteins are likely involved in the mechanism following light-dependent c-fosB induction. RNA interference (RNAi) of c-fosA/B significantly downregulated Fbxls expression, whereas RNAi of the Fbxl genes exerted no effect on c-fosB expression. The Fbxl genes showed rhythmic expression under light-dark cycles (LDs) with higher expression levels in early day (Fbxl16), whole day (Fbxl-like1), or day-to-early night (Fbxl4, Fbxl5, and Fbxl-like2), whereas their expression was reduced in the dark. We then examined the effect of their RNAi on the photic entrainment of the locomotor rhythm and found that RNAi of Fbxl4 either disrupted or significantly delayed the re-entrainment of the locomotor rhythm to shifted LDs. These results suggest that light-induced c-fosB expression stimulates Fbxl4 expression to reset the circadian clock.

Timeless Plays an Important Role in Compound Eye-Dependent Photic Entrainment of the Circadian Rhythm in the Cricket Gryllus bimaculatus.

The light cycle is the most powerful Zeitgeber entraining the circadian clock in most organisms. Insects use CRYPTOCHROMEs (CRYs) and/or the compound eye for the light perception necessary for photic entrainment. The molecular mechanism underlying CRY-dependent entrainment is well understood, while that of the compound eye-dependent entrainment remains to be elucidated. Using molecular and behavioral experiments, we investigated the role of timeless (tim) in the photic entrainment mechanism in the cricket Gryllus bimaculatus. RNA interference of tim (timRNAi) disrupted the entrainment or prolonged the transients for resynchronization to phase-delayed light-dark cycles. The treatment reduced the magnitude of phase delay caused by delayed light-off, but augmented advance shifts caused by light exposure at late night. TIM protein levels showed daily cycling with an increase during the night and reduction by light exposure at both early and late night. These results suggest that tim plays a critical role in the entrainment to delayed light cycles.

Nutrient conditions sensed by the reproductive organ during development optimize male fecundity in Drosophila.

Nutrient conditions affect the reproductive potential and lifespan of many organisms through the insulin signaling pathway. Although this is well characterized in female oogenesis, nutrient-dependent regulation of fertility/fecundity in males is not known. Seminal fluid components synthesized in the accessory gland are required for high fecundity in Drosophila males. The accessory gland is composed of two types of binucleated cells: a main cell and a secondary cell (SC). The transcription factors Defective proventriculus (Dve) and Abdominal-B (Abd-B) are strongly expressed in adult SCs, whose functions are essential for male fecundity. We found that gene expression of both Dve and Abd-B was down-regulated under nutrient-poor conditions. In addition, nutrient conditions during the pupal stage affected the size and number of SCs. These morphological changes clearly correlated with fecundity, suggesting that SCs act as nutrient sensors. Here, we provide evidence that Dve associates nutrient conditions with optimal reproductive potential in a target of rapamycin signaling-dependent manner.

The role of clockwork orange in the circadian clock of the cricket Gryllus bimaculatus.

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.