Loss of circadian clock function decreases reproductive fitness in males of Drosophila melanogaster.

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 approximately 40% 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.

Transplanted Drosophila excretory tubules maintain circadian clock cycling out of phase with the host.

Circadian rhythms in behaviors and physiological processes are driven by conserved molecular mechanisms involving the rhythmic expression of clock genes in the brains of animals [1]. The persistence of similar molecular rhythms in peripheral tissues in vitro [2] [3] suggests that these tissues contain self-sustained circadian clocks that may be linked to rhythmic physiological functions. It is not known how brain and peripheral clocks are organized into a synchronized timing system; however, it has been assumed that peripheral clocks submit to a master clock in the brain. To address this matter we examined the expression of two clock genes, period (per) and timeless (tim), in host and transplanted abdominal organs of Drosophila. We found that excretory organs in tissue culture display free-running, light-sensitive oscillations in per and tim gene activity indicating that they house self-sustained circadian clocks. To test for humoral factors, we monitored cycling of the TIM protein in excretory tubules transplanted into host flies entrained to an opposite light-dark cycle. We show that the clock protein in the donor tubules cycled out of phase with that in the host tubules, indicating that different organs may cycle independently, despite sharing the same hormonal milieu. We suggest that one way to achieve circadian coordination of physiological sub-systems in higher animals may be through the direct entrainment of light-sensitive clocks by environmental signals.

Rhythmic expression of a PER-reporter in the Malpighian tubules of decapitated Drosophila: evidence for a brain-independent circadian clock.

The protein product (PER) of the Drosophila clock gene, period (per), is involved in a molecular feedback loop in which PER inhibits the transcription of its own mRNA. This feedback causes the PER protein to cycle in a circadian manner, and this cycling in specific regions of the brain (the presumed location of the central pacemaker) is responsible for the rhythmicity of locomotor activity and possibly eclosion. PER has also been detected in several nonneural tissues in the abdomen, but whether PER exhibits free-running and light-sensitive cycles in any of these tissues is not known. In this study, the authors assayed the spatial and temporal distribution of a PER-reporter expressed in transgenic flies carrying a per-lacZ construct, which was shown to cycle in per-expressing brain cells. The authors demonstrate that this PER-reporter fusion protein cycles in the Malpighian tubules, showing first cytoplasmic accumulation, which is then followed by translocation of the signal into the nucleus. To test whether this rhythm was controlled by the brain, flies were decapitated and assayed for 3 days after decapitation. Expression patterns of PER-reporter in decapitated flies were nearly identical to those in intact flies reared in normal light-dark cycles, reversed light-dark cycles (phase shifted), and constant darkness. These results suggest that the Malpighian tubules contain a circadian pacemaker that functions independently of the brain.