Abstract
Pigment-dispersing factor neuropeptides (PDFs) occur in a wide range of protostomes including ecdysozoans (= molting animals) and lophotrochozoans (mollusks, annelids, flatworms, and allies). Studies in insects revealed that PDFs play a role as coupling factors of circadian pacemaker cells, thereby controlling rest-activity rhythms. While the last common ancestor of protostomes most likely possessed only one pdf gene, two pdf homologs, pdf-I and pdf-II, might have been present in the last common ancestors of Ecdysozoa and Panarthropoda (Onychophora + Tardigrada + Arthropoda). One of these homologs, however, was subsequently lost in the tardigrade and arthropod lineages followed by independent duplications of pdf-I in tardigrades and decapod crustaceans. Due to the ancestral set of two pdf genes, the study of PDFs and their receptor (PDFR) in Onychophora might reveal the ancient organization and function of the PDF/PDFR system in panarthropods. Therefore, we deorphanized the PDF receptor and generated specific antibodies to localize the two PDF peptides and their receptor in the onychophoran Euperipatoides rowelli. We further conducted bioluminescence resonance energy transfer (BRET) experiments on cultured human cells (HEK293T) using an Epac-based sensor (Epac-L) to examine cAMP responses in transfected cells and to reveal potential differences in the interaction of PDF-I and PDF-II with PDFR from E. rowelli. These data show that PDF-II has a tenfold higher potency than PDF-I as an activating ligand. Double immunolabeling revealed that both peptides are co-expressed in E. rowelli but their respective levels of expression differ between specific cells: some neurons express the same amount of both peptides, while others exhibit higher levels of either PDF-I or PDF-II. The detection of the onychophoran PDF receptor in cells that additionally express the two PDF peptides suggests autoreception, whereas spatial separation of PDFR- and PDF-expressing cells supports hormonal release of PDF into the hemolymph. This suggests a dual role of PDF peptides—as hormones and as neurotransmitters/neuromodulators—in Onychophora.
Highlights
Pigment-dispersing factors (PDFs) and pigment-dispersing hormones (PDHs) are neuropeptides that occur in various protostomes and have been mainly studied in nematodes, crustaceans and insects [reviewed in [1, 2]]
The initial BLAST search for PDF receptor homologs in three transcriptomes of E. rowelli yielded 27 candidate sequences. We included these in subsequent phylogenetic analyses of known bilaterian G protein-coupled receptors (GPCRs) to obtain the onychophoran PDFs and their receptor (PDFR) ortholog
We identified one PDF receptor gene (Er-pdfr) in each analyzed transcriptome of E. rowelli; only in one of the transcriptomes (ER10) the respective transcript was fractured in two contigs
Summary
Pigment-dispersing factors (PDFs) and pigment-dispersing hormones (PDHs) are neuropeptides that occur in various protostomes and have been mainly studied in nematodes, crustaceans and insects [reviewed in [1, 2]]. The PDFs of the fruit fly Drosophila melanogaster and the cockroach Rhyparobia maderae are analogs of the vasoactive intestinal peptide (VIP) of mammals [35]. It must be noted, that despite their similar function in the circadian pacemaker systems, the insect PDFs (and crustacean PDHs) do not share a common ancestry with VIP. While the last common ancestor of protostomes possessed only one pdf gene, which has been retained at least in mollusks and annelids [(36, 37); cf supporting information Supplementary Figures 1 and 2 in [38]], a duplication of this gene might have occurred in the ecdysozoan lineage (Figure 1). Even more intriguing is the independent duplication of the retained pdf-I gene in tardigrades, which show three in-paralogs, and decapod crustaceans, which express two to three PDH isoforms but seem to possess only two pdh genes [11, 38, 43,44,45,46,47,48]
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