Digging for Novel Neuropeptides: Identification and characterization of neuropeptides and their receptors in the earthworm, Dendrobaena veneta

About This Grant

Project Summary/Abstract Neuropeptides and peptide hormones orchestrate vital biological functions, including reproduction, feeding, and metabolism across all animal taxa. Studying these dynamic molecules and the genes that encode them across diverse species offers a powerful lens to understand the function of the nervous system. While most neuropeptides have been highly conserved throughout evolutionary history, some appear to have recently evolved in discrete taxa. Our first aim is to identify and characterize novel and classical neuropeptides and to annotate their conjugate receptors in the European Nightcrawler (Dendrobaena veneta) transcriptome. We have chosen to examine neuropeptides in an earthworm species because cell and molecular research on these animals has been neglected, and recent studies suggest that the genomes of this clade have undergone a massive and chaotic rearrangement. From these facts, we predict that we are likely to find previously uncharacterized modifications to neuropeptide signaling genes that inform our basic understanding of these molecules in earthworms. Our prediction has borne out: for proof of concept, we mined a short-read RNA- sequencing of earthworm epithelia and identified homologs to the neuropeptides myomodulin and FMRF- amide containing unique modifications, and one novel peptide. To complete our search for neuropeptides, we propose using long-read RNA-sequencing technology to produce high-fidelity transcriptomes of earthworm tissues enriched in signaling peptides and their receptors. In addition to using the standard tools for annotating de novo transcriptomes (Trinotate, PANNZER2, Funannotate, etc), undergraduate students will be trained in the writing of custom search algorithms. Our second aim is to characterize the physiological effect of earthworm neuropeptides by measuring the changes in the rhythmic contractions of earthworm’s digestive tract. In the earthworm crop-gizzard assay, changes in the rate and strength of contraction are used to assess the physiological properties of signaling molecules. Using this assay, our students have collected preliminary data demonstrating that the myomodulin peptide sequences we identified in Aim 1 have significant dosage- dependent effects on both the rate and force of crop-gizzard contractions. Our third aim is to identify the location and expression patterns of these neuropeptides and their conjugate receptors. Each segment of an earthworm contains a ganglia with neurons that innervate that segment. We will identify neuronal populations in the earthworm nervous system that release or respond to neuropeptides using immunofluorescence and in situ hybridization. Consistent with the R15 AREA program, undergraduate research assistants will be involved in every aspect of data collection and analysis, providing training opportunities in bioinformatics, physiology, and microscopy. Characterizing unique modifications in the neuropeptide signaling of evolutionary disparate species can pinpoint targets for the modulation of these molecules, which play an essential role in human physiology and disease.