Evolutionary and behavioral analysis of neuropeptides in bilaterians

Abstract

Neuropeptides are a diverse group of neurosecretory signaling molecules that are utilized by the nervous system of all bilaterian animals. These signaling molecules are involved in most physiological processes and can play major roles in animal behavior. Most of our knowledge about neuropeptides – whether in regards to neuropeptide diversity or the influence of neuropeptides on animal behavioral – originates from a collection of species that can be grouped into a few bilaterian clades. In this thesis, I investigate two different aspects of neuropeptide signaling, that involve animal species from clades that haven't been investigated before: the neuropeptide repertoire of xenacoelomorphs and the influence of neuropeptides on the behavior of planktonic larvae from a brachiopod and a nemertean species. A major bilaterian clade where nothing is known about the neuropeptide repertoire are the Xenacoelomorpha. According to recent phylogenetic analysis Xenacoelomorpha are the sister group to all remaining Bilateria (Deuterostomia + Protostomia) and therefore hold an important phylogenetic position for understanding bilaterian neuropeptide evolution. We identified the neuropeptide and neuropeptide receptor repertoire of xenacoelomorphs, by combining an in silico analysis of transcriptomes from 13 xenacoelomorph species with the mass spectrometric analysis of peptide extracts from 3 of these species. Our findings show the presence of several bilaterian neuropeptides as well as the presence of novel, xenacoelomorph specific neuropeptides and their diversification during xenacoelomorph evolution. Only a few functional studies have shown the influence of neuropeptides on the behavior of trochozoan larvae, and those used annelid or mollusc larvae. The only knowledge that we have about neuropeptidergic signaling in brachiopod or nemertean larvae originates from a few immunohistochemical studies. We investigated the excitatory peptide (EP) that has previously been identified in annelids and molluscs, where it showed myo-excitatory properties on tissue preparations of adult animals. We show that EP and the EP receptor are also present in brachiopods and nemerteans. We deorphanized the Lineus longissimus (Nemertea) EP receptor and show that EP can shift the swimming distribution of L. longissimus pilidium larvae in a water column upwards by increasing the beat frequency of the larval locomotory cilia. Another neuropeptide that we investigated is FLRFamide and its influence on the behavior of Terebratalia transversa (Brachiopoda) larvae. When mechanically disturbed, Terebratalia transversa larvae protrude their stiff and pointy chaetae in a defensive manner and sink down slowly. Both of these reactions can be induced simultaneously by FLRFamide. We deorphanized the T. transversa FLRFamide receptor and found its expression at the apical prototroch of the larvae and in the trunk musculature, which are the tissues that are responsible to perform the two sub-reactions. Customized antibodies against FLRFamide revealed FLRFamidergic nerves in and around the apical neuropil as well as FLRFamidergic nerves that directly innervate the trunk musculature. In this thesis, I present a substantial dataset of neuropeptides and neuropeptide receptors from different species of the Xenacoelomorpha, an animal clade where nothing has known about their neuropeptide repertoire before. I also present two studies where I show how neuropeptides can influence the behavior of brachiopod and nemertean planktonic larvae.