Circadian coordination of life functions is believed to contribute to an organism's fitness; however, such contributions have not been convincingly demonstrated in any animal. The most significant measure of fitness is the reproductive output of the individual and species. Here we examined the consequences of loss of clock function on reproductive fitness in Drosophila melanogaster with mutated period (per 0 ), timeless (tim 0 ), cycle (cyc 0 ), and Clock (Clk Jrk ) genes. Single mating among couples with clock-deficient phenotypes resulted in Ϸ40% fewer progeny compared with wild-type flies, because of a decreased number of eggs laid and a greater rate of unfertilized eggs. Male contribution to this phenotype was demonstrated by a decrease in reproductive capacity among per 0 and tim 0 males mated with wild-type females. The important role of clock genes for reproductive fitness was confirmed by reversal of the low-fertility phenotype in flies with rescued per or tim function. Males lacking a functional clock showed a significant decline in the quantity of sperm released from the testes to seminal vesicles, and these tissues displayed rhythmic and autonomous expression of clock genes. By combining molecular and physiological approaches, we identified a circadian clock in the reproductive system and defined its role in the sperm release that promotes reproductive fitness in D. melanogaster.M any life functions, from cellular activities to behavior, display daily (circadian) rhythms. These rhythms are generated by cell-autonomous circadian clocks and involve several genes encoding transcriptional regulators, which are substantially conserved among animals ranging from fruit flies to humans (1). The core clock mechanism in Drosophila melanogaster involves rhythmic transcription of the period (per) and timeless (tim) genes followed by nuclear accumulation of their proteins, PER and TIM. Another gene, Clock (Clk), shows mRNA oscillations that are out of phase with the oscillations of per and tim mRNAs. Protein encoded by Clk, together with protein encoded by the gene cycle (cyc), activates transcription of per and tim. These four genes are essential for the function of the brain clock, and a null mutation in any of them renders the fly behaviorally arrhythmic (2).Although the importance of circadian clocks in controlling behavior is well documented, their functional significance for physiology is less understood. Clock genes are rhythmically and autonomously expressed in peripheral tissues of D. melanogaster (3-5), zebrafish (6), and mammals (7), suggesting that peripheral clocks may coordinate physiological processes ultimately affecting fitness. Few studies have compared physiological parameters between individuals with a normal or disrupted circadian clock in any species. A study in cyanobacteria demonstrated that colonies in which free-running circadian periods are in resonance with environmental 12-h light͞12-h dark cycles (LD) out-compete colonies in which internal and external periodicities are out of synchro...

Circadian clock genes are ubiquitously expressed in the nervous system and peripheral tissues of complex animals. While clock genes in the brain are essential for behavioral rhythms, the physiological roles of these genes in the periphery are not well understood. Constitutive expression of the clock gene period was reported in the ovaries of Drosophila melanogaster; however, its molecular interactions and functional significance remained unknown. This study demonstrates that period (per) and timeless (tim) are involved in a novel noncircadian function in the ovary. PER and TIM are constantly expressed in the follicle cells enveloping young oocytes. Genetic evidence suggests that PER and TIM interact in these cells, yet they do not translocate to the nucleus. The levels of TIM and PER in the ovary are affected neither by light nor by the lack of clock-positive elements Clock (Clk) and cycle (cyc). Taken together, these data suggest that per and tim are regulated differently in follicle cells than in clock cells. Experimental evidence suggests that a novel fitness-related phenotype may be linked to noncircadian expression of clock genes in the ovaries. Mated females lacking either per or tim show nearly a 50% decline in progeny, and virgin females show a similar decline in the production of mature oocytes. Disruption of circadian mechanism by either the depletion of TIM via constant light treatment or continuous expression of PER via GAL4/UAS expression system has no adverse effect on the production of mature oocytes.

Release of sperm bundles from moth testes is controlled by the local circadian oscillator. The mechanism which restricts migration of sperm bundles to a few hours each day is not understood. We demonstrate that a daily cycle of sperm release is initiated by the migration of folded apyrene sperm bundles through a cellular barrier at the testis base. These bundles have conspicuous concentrations of actin filaments at their proximal end. Inhibition of actin polymerization by cytochalasin at aspecific time of day inhibited sperm release from the testis. Likewise, application of double-stranded actin RNA specifically inhibited sperm release. This RNA-mediated interference (RNAi) lowered the pool of actin mRNA in tissues involved in sperm release. The decline in mRNA levels resulted in the selective depletion of F-actin from the tip of apyrene sperm bundles, suggesting that this actin may be involved in the initiation of sperm release. Combined results of RNAi experiments at physiological, cellular and molecular levels identified unique cells that are critically involved in the mechanism of sperm release.