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Pigment-dispersing factor PDF is an 18-amino acid insect neuropeptide that mediates a circadian rhythmicity in locomotor activity. PDF is coded in a precursor protein together with another neuropeptide named PDF-associated peptide, PAP. PDF is highly conserved among insects, whereas the homology of PAPs is very low with considerably varied amino acid sequences. Since such dissimilarity has suggested that the function of PAP peptide is not associated with that of PDF, we have attempted to analyze the sequences of PDF precursor proteins among a series of species of insects and hypothesized that PDF precursors are classified into at least three different classes: Drosophila-Musca, Meimuna-Romalea, and Gryllus. In order to exemplify this hypothesis, we here describe the molecular cloning of the pdf-gene of the black blowfly Phormia regina and an in silico screening for the pdf-gene in the genome databank of the mosquito Anopheles gambie, both species belonging to the Diptera. It was found that deduced amino acid sequences of PDF peptides are almost completely conserved among all Dipterans and also the amino acid sequences of PAPs are considerably highly preserved (55–82 similarity) among the species of Diptera. The results confirmed the validity of grouping the PDF precursor proteins. In situ hybridization was carried out in fly brains to identify the precise locations of pdf-expressing cells and to examine any daily cycling of pdf mRNA. Intense signals for pdf mRNA were identified in the medulla, but not in the pars lateralis where PDF neurons were strongly immunostained by the antibody raised against PDF peptide. Hybridization was also performed for the brain samples at two hour intervals throughout the day. Although very intense hybridization signals were observed at ZT8 even in some neurites, no prominent rhythmicity of pdf mRNA expression was observed.

Pigment-dispersing factor (PDF), an 18-amino acid neuropeptide, is a principal circadian neurotransmitter for the circadian rhythms of the locomotor activity in flies. Recently, two completely different types of PDF precursor were clarified; that of the cricket Gryllus bimaculatus and that of the last-summer cicada Meimuna opalifera. The G. bimaculatus PDF precursor is extraordinarily short and comprises a nuclear localization signal (NLS), while the M. opalifera PDF precursor is of ordinary length, comparable to that seen for the precursors of crustacean β-PDH homologues. Although their PDF peptide regions were exactly the same, the regions containing a signal peptide combined with a PDF-associated peptide (PAP) were remarkably different from each other. Such a grouping suggested a fundamental role for the PAP peptide in the circadian clock, perhaps associated with PDF function. In the present study, the cDNA cloning of PDF from the adult brains of the housefly Musca domestica was carried out and it was found that an isolated clone (527 bp) encodes a PDF precursor protein of ordinary length. The PDF peptide shows a high sequence identity (78%–94%) and similarity (89%–100%) to insect PDFs and also to the crustacean β-PDH peptides. In particular, there is only a single amino acid difference between the PDFs of Musca and Drosophila; at position 14 Ser for Musca PDF and Asn for Drosophila PDF. A characteristic Ser10 in Drosophila was retained in Musca, indicating the presence of a structural profile unique to these PDFs. The results of sequence analyses suggest that Musca and Drosophila PDFs are to be considered members of a single group that has evolved structurally. When the primary structure of the PAP regions was compared, the Musca PDF precursor also belonged to the same group as that to which the Drosophila PDF precursor belongs. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd.

Pigment-dispersing factor PDF is an 18-amino acid insect neuropeptide that mediates a circadian rhythmicity in locomotor activity. PDF is coded in a precursor protein together with another neuropeptide named PDF-associated peptide, PAP. PDF is highly conserved among insects, whereas the homology of PAPs is very low with considerably varied amino acid sequences. Since such dissimilarity has suggested that the function of PAP peptide is not associated with that of PDF, we have attempted to analyze the sequences of PDF precursor proteins among a series of species of insects and hypothesized that PDF precursors are classified into at least three different classes: Drosophila-Musca, Meimuna-Romalea, and Gryllus. In order to exemplify this hypothesis, we here describe the molecular cloning of the pdf-gene of the black blowfly Phormia regina and an in silico screening for the pdf-gene in the genome databank of the mosquito Anopheles gambie, both species belonging to the Diptera. It was found that deduced amino acid sequences of PDF peptides are almost completely conserved among all Dipterans and also the amino acid sequences of PAPs are considerably highly preserved (55–82 similarity) among the species of Diptera. The results confirmed the validity of grouping the PDF precursor proteins. In situ hybridization was carried out in fly brains to identify the precise locations of pdf-expressing cells and to examine any daily cycling of pdf mRNA. Intense signals for pdf mRNA were identified in the medulla, but not in the pars lateralis where PDF neurons were strongly immunostained by the antibody raised against PDF peptide. Hybridization was also performed for the brain samples at two hour intervals throughout the day. Although very intense hybridization signals were observed at ZT8 even in some neurites, no prominent rhythmicity of pdf mRNA expression was observed.

Many lines of evidence have suggested that neuropeptides other than pigment-dispersing factor (PDF) are involved in regulating insect circadian rhythms, and FMRFamide-related peptides are additional candidates acting as such neuromodulators. Double-immunolabelling in insect brains with anti-crustacean beta-PDH and anti-FMRFamide antibodies had previously suggested that insect PDF and FMRFamide-like peptides may coexist in the same cells. However, it is critical for this kind of comparative investigations to use antibodies of proven specificity, to eliminate the possibility of both reciprocal cross-reactivity and the detection of unknown peptides. In the present study, we achieved the cDNA cloning of an fmrf mRNA from the housefly Musca domestica, for which co-localization of FMRFamide and PDF peptides was previously suggested. In order to examine the possible co-expression of this gene with the pdf gene, we carried out double-labelled in situ hybridization for simultaneous detection of both pdf and fmrf mRNAs in housefly, Musca brains. The results clearly indicated that they occur in distinctly different cells. This was also proven for the fruit fly Drosophila melanogaster by similar double-labelled in situ hybridization. The results thus revealed no reason to evoke the physiological release of FMRFamide and PDF peptides from the same neurons.