Molecular cloning and circadian expression profile of insect neuropeptide PDF in black blowfly, Phormia regina

Abstract

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, andGryllus. In order to exemplify this hypothesis, we here describe the molecular cloning of thepdf-gene of the black blowflyPhormia regina and anin silico screening for thepdf-gene in the genome databank of the mosquitoAnopheles 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 ofpdf-expressing cells and to examine any daily cycling ofpdf mRNA. Intense signals forpdf 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 ofpdf mRNA expression was observed.