Analysis of Pigment-Dispersing Factor Neuropeptides and Their Receptor in a Velvet Worm

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.

The velvet worm brain unveils homologies and evolutionary novelties across panarthropods

“…Note that correspondent terms do not necessarily imply homology with homonymous structures in arthropods and other animals. Whenever possible, the nomenclature follows the terms and definitions suggested by Ito et al [49] for insects and Richter et al [20] Antennal nerve [15,32,34,40,43,48,[54][55][56][57][58][59][60][61][62][63][64][65][66][67][68] Antennal neuropil [57] Antennal tract [18,32,38,39,43,47,48,[69][70][71][72] Antennalstrang [German] [27,28,30,31,51] Antennary nerve [25,[73][74][75][76] Antennennerv [German] [73,74] Anterior appendage tract [13] Dorsolate...…”

“…nerf antennaire [French] [26,77] Nerve strand of the antenna [65] nervo antenna [Portuguese] [78] Nervus antennalis [Latin] [30,53,79] Tentakelnerv [German] [80] Antennal neuropil [ Antennal lobe [32,34,39,43,47,48,54,56,65,69] Antennal nerve [15,34,40,48,55,[57][58][59][66][67][68] Antennal nerve cord [33] Antennalstrang [German] [27,28,30,31,51] Antennal tract [18,34,38,39,43,47,56,59,[69][70][71][72] Antennennerv [German] [52] Anterior appendage tract …”

“…Note that correspondent terms do not necessarily imply homology with homonymous structures in arthropods and other animals. Whenever possible, the nomenclature follows the terms and definitions suggested by Ito et al [49] for insects and Richter et al [20] for invertebrates in general (Continued) Central complex [75] corpo central [Portuguese] [78] corpo estriado [Portuguese] [78] corpus centrale [Latin] [43] corpus striatum [Latin] [32,54,56] Corpus striatum [Latin] [27,28,30] dorsal cap [90] gestreifter Körper [German] [27,28,30] Posterodorsal lobe [25] Zentralkörper Central fibrous mass [56] Central lobe [25,81,82] Median lobe [25] Zentralfasermasse Circumpharyngeal connective [92] Circumpharyngeal medullary connective [68] commissure oesophagienne [French] [26] Connecting cord [38,39,55,69,86] Connecting piece [40,48,72] Connective of the brain [93] Connective to the ventral nerve cord [65] Table 1 Glossary of neuroanatomical terms for studies of Onychophora. Terms that are highlighted in bold and preceded by arrows are defined in corresponding fields.…”

“…Finally, previous investigations of the onychophoran brain suggest that various structures remain to be described and characterised (e.g. an "anterior neuropil" referred to in [38,48]), and emphasises that current understanding of the onychophoran brain is far from complete. Furthermore, the hitherto known architecture of the onychophoran brain has not been critically reappraised, although some features have already been questioned (e.g.…”

“…Accordingly, the range of techniques used to investigate the onychophoran nervous system is largely limited to classical methods. Furthermore, most studies have focussed on specific aspects of the nervous system, such as neurogenesis and neural development [13,15,[34][35][36][37] or on the localisation of specific structures and neurons using selective markers [14,18,33,[38][39][40][41][42]. Thus, a comprehensive overall picture is still lacking, especially in the light of the array of new methods currently available.…”