Structural isoforms of the circadian neuropeptide PDF expressed in the optic lobes of the cricket Gryllus bimaculatus: Immunocytochemical evidence from specific monoclonal antibodies

Abstract

Pigment-dispersing factor (PDF) is an 18-mer peptide that acts as a principal neurotransmitter of the insect circadian clock. Our previous study, utilizing anti-Uca beta-PDH polyclonal antibody (pAb) to immunolabel the optic lobe of the cricket Gryllus bimaculatus, suggested the existence of an alternative PDF-like peptide in the outer cells of the first neuropile, or lamina (La), which were much less immunoreactive than the inner cells of the second neuropile, the medulla (Me). To obtain structural information about such a PDF-like peptide, we prepared 10 anti-Gryllus PDF monoclonal (mAb) and pAb antibodies and analyzed their detailed epitope specificities. The PDFMe and PDFLa inner cells and their axonal projections were clearly immunoreactive to all these antibodies, revealing the widespread immunocytochemical organization of the PDF system in the optic lobe, as seen previously with anti-Uca beta-PDH pAb and anti-Gryllus PDF mAb, the epitope structures of which were also clarified in this study. The lamina outer cells, which we found lacked a target pdf mRNA, displayed specific immunoreactivities, indicating that the cells contain a distinct PDF-like peptide possessing both N- and C-terminal structures. These cells were not immunolabeled by some other monoclonal antibodies, however, implying that the PDFLa outer cells have a PDF isoform peptide devoid of Asn at positions 6 and 16. This isoform was also identified in a varicose arborization in the lamina. These results suggest not only the structure of the peptide, but also the possibility of additional functions of this novel PDF isoform.

Pigment-dispersing hormone in Daphnia interneurons, one type homologous to insect clock neurons displaying circadian rhythmicity

“…10). This was most likely the reason for the clear detection of Daphnia PDH with our polyclonal antibodies against beta-type Uca-PDH that recognize a common epitope in the first 12aas of many decapod PDHs and insect PDFs [4,5,46]. However, among the last 6aas almost all are unusual except Met 15 , which renders the peptide susceptible to sulfoxidation.…”

“…The fragmentation patterns were analyzed by use of the Data ExplorerT software 4.3 package to manually determine the sequence of the peptide. Antisera, immunocytochemistry, and controls A rabbit antiserum against synthetic Uca-PDH (= beta-PDH, [5]; code 3B3) was used that has been scrutinously characterized for epitope specificity in several previous studies on crustaceans and insects [5,45,46]. For indirect immunofluorescence (IIF) staining of whole-mount preparations, tissues were washed in 0.01 M sodium phosphate buffer, pH 7.4, containing 0.9% NaCl and 0.02% sodium azide (PTXN) and incubated with anti-PDH-serum diluted 1:16,000 to 1:24,000 overnight at RT followed by fluorescein-isothiocyanate-labeled goat anti-rabbit IgG (FITC-GAR, Sigma, Taufkirchen, Germany) diluted 1:50-1:80 in PTXN for 1 h in darkness at RT.…”

Circadian pacemaker coupling by multi-peptidergic neurons in the cockroach Leucophaea maderae

“…3B3) was a gift from Dr. Heinrich Dircksen, Stockholm and was raised in rabbits against synthetic Uca pugilator β-PDH (Dircksen et al 1987). This antiserum has been widely used to detect PDF-expressing neurons in many insect species and has recently been systematically characterized by Honda et al (2006). Furthermore, a PDF peptide was previously isolated in L. maderae by Hamasaka et al (2005).…”

Roles of PER immunoreactive neurons in circadian rhythms and photoperiodism in the blow fly, Protophormia terraenovae

“…The rabbit anti-Gryllus bimaculatus-PDF antiserum was provided by Dr K. Tomioka (Okayama University, Okayama, Japan). The epitope structures recognized by the anti-Gryllus bimaculatus-PDF have been well characterized by enzyme-linked immunosorbent assay (Honda et al, 2006). The sections were incubated in the anti-PDF antiserum at a dilution of 1:5000 for 1 day at 4°C.…”