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The clock network of Drosophila melanogaster expresses various neuropeptides, but a function in clock-mediated behavioral control was so far only found for the neuropeptide pigment dispersing factor (PDF). Here, we propose a role in the control of behavioral rhythms for the ion transport peptide (ITP), which is expressed in the fifth small ventral lateral neuron, one dorsal lateral neuron, and in only a few nonclock cells in the brain. Immunocytochemical analyses revealed that ITP, like PDF, is most probably released in a rhythmic manner at projection terminals in the dorsal protocerebrum. This rhythm continues under constant dark conditions, indicating that ITP release is clock controlled. ITP expression is reduced in the hypomorph mutant ClkAR, suggesting that ITP expression is regulated by CLOCK. Using a genetically encoded RNAi construct, we knocked down ITP in the two clock cells and found that these flies show reduced evening activity and increased nocturnal activity. Overexpression of ITP with two independent timeless-GAL4 lines completely disrupted behavioral rhythms, but only slightly dampened PER cycling in important pacemaker neurons, suggesting a role for ITP in clock output pathways rather than in the communication within the clock network. Simultaneous knockdown (KD) of ITP and PDF made the flies hyperactive and almost completely arrhythmic under constant conditions. Under light-dark conditions, the double-KD combined the behavioral characteristics of the single-KD flies. In addition, it reduced the flies' sleep. We conclude that ITP and PDF are the clock's main output signals that cooperate in controlling the flies' activity rhythms.

Comparative studies on cellular and molecular clock mechanisms have revealed striking similarities in the organization of the clocks among different animal groups. To gain evolutionary insight into the properties of the clock network within the Drosophila genus, we analyzed sequence identities and similarities of clock protein homologues and immunostained brains of 10 different Drosophila species using antibodies against vrille (VRI), PAR-protein domain1 (PDP1), and cryptochrome (CRY). We found that the clock network of both subgenera Sophophora and Drosophila consists of all lateral and dorsal clock neuron clusters that were previously described in Drosophila melanogaster. Immunostaining against CRY and the neuropeptide pigment-dispersing factor (PDF), however, revealed species-specific differences. All species of the Drosophila subgenus and D. pseudoobscura of the Sophophora subgenus completely lacked CRY in the large ventrolateral clock neurons (lLNvs) and showed reduced PDF immunostaining in the small ventrolateral clock neurons (sLNvs). In contrast, we found the expression of the ion transport peptide (ITP) to be consistent within the fifth sLNv and one dorsolateral clock neuron (LNd) in all investigated species, suggesting a conserved putative function of this neuropeptide in the clock. We conclude that the general anatomy of the clock network is highly conserved throughout the Drosophila genus, although there is variation in PDF and CRY expression. Our comparative study is a first step toward understanding the organization of the circadian clock in Drosophila species adapted to different habitats., AuthorCount:7