To determine the in vivo regulatory pattern of the clock gene period (per), the authors recently developed transgenic Drosophila carrying a luciferase cDNA fused to the promoter region of per. They have now carried out noninvasive, high time-resolution experiments allowing high-throughput monitoring of circadian bioluminescence rhythms in individual living adults for several days. This immediately solved several problems (resulting directly from individual asyn chrony within a population) that have accompanied previous biochemical ex periments in which groups of animals were sacrificed at each time point. Furthermore, the authors have developed numerical analysis methods for auto matically determining rhythmicity associated with bioluminescence records from single flies. This has revealed some features of per gene transcription that were previously unappreciated and provides a general strategy for the analysis of rhythmic time series in the study of molecular rhythms.

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 ≈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 .

The neuropeptide pigment-dispersing factor (PDF) is a key transmitter in the circadian clock ofDrosophila melanogaster.PDF is necessary for robust activity rhythms and is thought to couple the circadian oscillations of the clock neurons. However, little is known about the action of PDF on individual clock neurons. Here, we combined theperiod–luciferase reporter system with immunolabeling of clock proteins in wild-type andPdf⁰¹mutants to dissect the effects of PDF on specific subgroups of clock neurons. Additionally, PDF levels were elevated to higher than normal levels using specific neural mutants, and a correlation analysis of locomotor activity and clock protein staining served to determine the periods of specific clock cells. We found that PDF has multiple effects on the clock neurons: In some groups of clock neurons, PDF was required for maintaining the oscillations of individual cells, and in others, PDF was required for synchronous cycling of the individual members. Other clock neurons cycled with high amplitude in absence of PDF, but PDF affected their intrinsic clock speed. Sometimes PDF shortened and sometimes PDF lengthened period. Our observations indicate that PDF is crucial for adjusting cycling amplitude, period, and phase of the different players in the circadian clock. Under natural conditions PDF may be required for adaptingDrosophila's clock to varying photoperiods. Indeed, we show here thatPdf⁰¹mutants are not able to adapt their activity to long photoperiods in a wild-type manner.