Nemertean, Brachiopod, and Phoronid Neuropeptidomics Reveals Ancestral Spiralian Signaling Systems

Abstract

Neuropeptides are diverse signaling molecules in animals commonly acting through G-protein coupled receptors (GPCRs). Neuropeptides and their receptors underwent extensive diversification in bilaterians and the relationships of many peptide-receptor systems have been clarified. However, we lack a detailed picture of neuropeptide evolution in lophotrochozoans as in-depth studies only exist for mollusks and annelids. Here, we analyze peptidergic systems in Nemertea, Brachiopoda, and Phoronida. We screened transcriptomes from 13 nemertean, 6 brachiopod, and 4 phoronid species for proneuropeptides and neuropeptide GPCRs. With mass spectrometry from the nemertean Lineus longissimus, we validated several predicted peptides and identified novel ones. Molecular phylogeny combined with peptide-sequence and gene-structure comparisons allowed us to comprehensively map spiralian neuropeptide evolution. We found most mollusk and annelid peptidergic systems also in nemerteans, brachiopods, and phoronids. We uncovered previously hidden relationships including the orthologies of spiralian CCWamides to arthropod agatoxin-like peptides and of mollusk APGWamides to RGWamides from annelids, with ortholog systems in nemerteans, brachiopods, and phoronids. We found that pleurin neuropeptides previously only found in mollusks are also present in nemerteans and brachiopods. We also identified cases of gene family duplications and losses. These include a protostome-specific expansion of RFamide/Wamide signaling, a spiralian expansion of GnRH-related peptides, and duplications of vasopressin/oxytocin before the divergence of brachiopods, phoronids, and nemerteans. This analysis expands our knowledge of peptidergic signaling in spiralians and other protostomes. Our annotated data set of nearly 1,300 proneuropeptide sequences and 600 GPCRs presents a useful resource for further studies of neuropeptide signaling.

Keywords: Key words: RFamide; APGWamide; GPCRs; GPR139; GnRH; Trochozoa; agatoxin-like peptide; neuropeptide; pleurin; vasopressin.

Fig. 1.

Investigated taxa and pipeline for the identification of peptidergic signaling systems. (A) Investigated taxa. Numbers in square brackets indicate the number of identified neuropeptide precursor types (out of a total of 72 types), number of identified neuropeptide GPCR types (out of a total of 41 known types), and BUSCO completeness of transcriptomes (in %). The depicted relationships of nemertean species are based on Andrade et al. (2014) and Kvist et al. (2015), the relationships of phoronids are based on Santagata and Cohen (2009), the relationship of brachiopod species is based on Kocot et al. (2017) and Marlétaz et al. (2019), with the taxonomic classifications according to http://www.marinespecies.org/ (status: July 2020; last accessed July 20, 2021). (B) Pipeline for the identification of proneuropeptides and neuropeptide GPCRs. (C) Scanning electron micrograph of a Lineus longissimus (Nemertea) larva. (D) SEM image of a Terebratalia transversa (Brachiopoda) larva. (E) SEM image of a Phoronis muelleri (Phoronida) larva. Scale bars: 50 µm.

Fig. 2.

Cluster analysis of neuropeptide precursors. Connections are based on blast similarities <1e-10 as shown on the upper right. Animal groups are color and symbol coded as shown on the upper left. 7B2, Neuroendocrine protein 7B2; a, amide; ALP, agatoxin-like peptide; AKH, adipokinetic hormone; Asta, allatostatin; Bur, Bursicon; CCAP, crustacean cardioacceleratory peptide; CP3-r, CCWamide-Prohormone 3-related; CRF, corticotropin releasing factor; DH, diuretic hormone; ELH, egg-laying hormone; EP, excitatory peptide; ETH, ecdysis triggering hormone; GlyHo-A, glycoprotein hormone alpha; GlyHo-B, glycoprotein hormone beta; GnRH, gonadotropin-releasing hormone; ILGFBP, insulin-like growth factor binding protein; L11, elevenin; Leucok, leucokinin; MIP, myoinhibitory peptide/allatostin B; NpY/F, neuropeptide Y/F; PBAN, pheromone biosynthesis activating neuropeptide; PDF, pigment dispersing factor; PP, pedal peptide; ProHo, Prohormone; PTTH, Prothoracicotropic hormone; RFa’s, RFamides, Wa’s, Wamides.

Fig. 3.

Neuropeptides discovered by mass spectrometry in Lineus longissimus. Spectra of newly identified peptides and their precursor sequences. (A) WWS peptide. (B) DMF peptide. (C) AGEamide. (D) GGRWamide. (E) GxGH peptide. Spectra of peptides are shown on the left side of the panels with the corresponding precursor sequences shown to the right. Precursor sequences are marked as follows: signal peptide in blue, detected peptide in bold, name-giving sequence underlined, cleavage sites in magenta, cysteine residues in green. The precursor of MS peptide 4 (GGRWamide) is split into two partial sequences.

Fig. 4.

Maximum-likelihood analysis of rhodopsin and secretin-type neuropeptide GPCRs. (A) Rhodopsin beta GPCRs. (B) Rhodopsin gamma GPCRs. (C) Secretin GCPRs. Terminal branches are color-coded according to taxon as shown on the upper left. SH-aLRT support values of major nodes are color-coded in circles as indicated on the lower left. Scale bars on the lower right of each tree indicate the inferred amino acid substitutions per site. Dashed lines demarcate orthologous receptor types. A double crossing through a branch (nemertean FMRFamide type 1 receptors) indicates that the branch length was halfened. AKH, adipokinetic hormone; Asta, allatostatin; CCAP, crustacean cardioacceleratory peptide; CCK, cholecystokinin; CRF, corticotropin releasing factor; DH, diuretic hormone; ETH, ecdysis triggering hormone; EP, excitatory peptide; GCG, glucagon, GHS, growth hormone secretagogue; GnRH, gonadotropin releasing hormone; GRP, gastrin releasing peptide; iPTH, insect parathyroid hormone; L-DCc, lophotrochozoan DH31/Calcitonin cluster; NMU, neuromedin-U; NPS, neuropeptide-S; NPY/NPF, neuropeptide Y, neuropeptide F; MCH, melanin concentrating hormone; MIP, myoinhibitory peptide; PACAP, pituitary adenylate cyclase-activating polypeptide; PBAN, pheromone biosynthesis activating neuropeptide; PDF, pigment dispersing factor; pQRFPa, pyroglutamylated RFamide peptide; PTH, parathyroid hormone; PRP, prolactin releasing peptide; SH, Shimodaira–Hasegawa approximate-likelihood ratio test; TRH, thyrotropin releasing hormone; VIP, vasoactive intestinal peptide.

Fig. 5.

Presence of proneuropeptides and neuropeptide GPCRs. Signaling systems are divided into monophyletic GPCR groups in agreement with previous studies (Elphick et al. 2018; Thiel et al. 2018), with the exception of the rhodopsin beta type GPCRs with unstable phylogenetic positions. The presented resolution goes back to the last common ancestor of the five phyla shown, although many of the groups have deeper conservation in bilateria as also evident from the trees in figure 4. A filled circle indicates the presence of a propeptide in at least one taxon of the corresponding clade, a filled square around the lower half of the circle indicates the presence of a receptor. A white circle or square with full line indicates that the precursor or receptor was not found in any of the species of this animal group, but potential orthologs are known from other lophotrochozoans. If the circle or square has a dotted line, the corresponding precursor or receptor is generally not known in lophotrochozoans. The QSGamide/iPTH peptide–receptor pairing is assumed based on the QSGamide and iPTH orthology (Xie et al. 2020) but has not been proven in Lophotrochozoa. *The presence of leucine-rich-repeat containing GPCRs or non-GPCR neuropeptide receptors was not investigated in this study. **F[V/L/I]amides may include phylogenetically different spiralian peptides with similar C-terminal motifs. ***The depicted NKY receptors and short neuropeptide F receptors refer to the same receptor. lccr, leucine-rich repeat containing. Peptides and receptors are paired according to Jékely (2013), Bauknecht and Jékely (2015), Elphick et al. (2018), Schwartz et al. (2019), and Xie et al. (2020).

Fig. 6.

Peptide alignments and genomic precursor structure. (A) Alignment of CCWamide and agatoxin-like peptides. (B) Genomic exon–intron structure of CCWamide and agatoxin-like peptide precursors. (C) Alignment of pleurin peptides. (D) Genomic exon–intron structure of pleurin precursors. (E) Genomic exon–intron structure of APGWamide and RGWamide precursors. [An] annelid, [Ar] arthropod, [Br] brachiopod, [Mo] mollusk, [Ne] nemertean, [Ph] phoronid.

Fig. 7.

Vasotocin-related and GnRH-related peptides. (A) Precursor structure of L. longissimus vasotocin-related paralogs. Length of signal peptide in amino acids is shown above the precursors, rest is to scale. (B) Alignment of vasotocin-related peptides. (C) Evolution of vasotocin-related proneuropeptides in nemerteans. Precursors are not to scale. (D) Alignment of GnRH and related peptides. B. mori, Bombyx mori; C. gigas, Crassostrea gigas; Nrph, neurophysin; p1, paralog 1; p2, paralog 2; Pdu, Platynereis dumerilii; [An], annelid; [Ar], arthropod; [Br], brachiopod; [Mo], mollusk; [Ne], nemertean; [Ph], phoronid.