Chronobiological analysis and mass spectrometric characterization of pigment-dispersing factor in the cockroach Leucophaea maderae

Pigment-dispersing factor (PDF) is a neuropeptide that plays a prominent role in the circadian clock of several insects. The cockroach Leucophaea maderae was the first animal where the site of a biological clock could be located, and still is a focal point of circadian research. Although detailed studies on the action of pigment-dispersing factor and the distribution of PDF-like immunoreactivity in the L. maderae brain exist, a native pigment-dispersing factor of this species has not been characterized so far. The authentic Lem-PDF was isolated from L. maderae by a combination of high performance liquid chromatography, crab pigment-dispersion bioassay and an immunosorbent assay. Mass spectrometric characterization and the conserved sequence of pigment-dispersing factor in orthopteromorphan insects suggest that Lem-PDF has the sequence NSEXINSLLGLPKVLNDAa (where X= I or L). Lem-PDF is thus identical to either Periplaneta americana PDF or Acheta domesticus PDF. Detailed analysis of PDF-like immunofluorescence in different regions of the brain suggests that there are no drastic daily changes in the amount of pigment-dispersing factor as occur in Drosophila melanogaster, which might be explained by a lack of circadian pigment-dispersing factor release and production, or by phase differences between the pigment-dispersing factor neurons. Abbreviations ESI-Q-TOF MS electrospray ionization-quadrupole-time of flight mass spectrometry. MALDI-TOF MS matrix-assisted laser desorption ionization-time of flight mass spectrometry. PDF pigment-dispersing factor (Drm-PDF Drosophila melanogaster-PDF; Lem-PDF, Leucophaea maderae-PDF; Pea-PDF, Periplaneta americana-PDF). PDH pigment-dispersing hormone; s-LN(v)s and lLN(v)s small and large ventral lateral neurons. ZT Zeitgeber time;