Overview of the neuropeptide precursors in U. unicinctus
We performed BLAST search and NpSearch prediction to screen the neuropeptide precursors in the transcriptomes of U. unicinctus. A total of 54 neuropeptide precursors (pNPs) were identified, 7 from BLAST search, 5 from NpSearch prediction, and 42 from both methodologies (Fig. 1A and Supplementary Table S2). Among them, 49 pNPs had been reported in other species, and the remaining 5 pNPs were first identified in U. unicinctus and we named them FxFamide, FILamide, FW, FRWamide and ASYY according to their conserved amino acid residues. In the U. unicinctus transcriptomes, most neuropeptide precursor sequences contained the full-length open reading frame (ORF) with a signal peptide (SP), except pedal peptide 1 and FVRIamide. The sequence characteristics of U. unicinctus neuropeptide precursors for the SP presence, the conserved peptide motifs and other hallmarks of bioactive peptides, e.g. amidation C-terminal Gly, Cys-containing stretches, mono- or dibasic cleavage sites were summarized in Fig. 1A and Supplementary Fig. S1.
Due to the inherent difficulties of analyzing highly diverse and repetitive pNPs, the relationships among different families are often elusive. Therefore, Jékely [28] and Conzelmann [29], using similarity-based clustering and sensitive similarity searches, obtained a global view of metazoan pNP diversity and evolution based on a curated dataset of 6,225 pNPs from 10 phyla. This approach was also useful for analyzing the phylogenetic distribution of U. unicinctus pNPs and we classified the pNP families using the same methodology. The results showed that ten pNPs in U. unicinctus were categorized as the ancient eumetazoan families (Fig. 1A), which are the repertoire neuropeptides with the short amidated peptides, such as R[F/Y]amide, Wamide, insulin-related peptide and the glycoprotein hormones [28, 30, 31]. Then, twenty-four pNPs in U. unicinctus were categorized as the ancient bilaterian families (Fig. 1A), which belong to 17 neuropeptide families [28]. Three members of the ancient protostome neuropeptide precursor families were present in U. unicinctus, including prohormone-3, myomodulin, and whitnin-2 (Fig. 1A). Moreover, we identified nine pNPs in U. unicinctus (Fig. 1A), which were proposed to be the lophotrochozoan-specific families [29]. Three pNPs in U. unicinctus had recognizable orthologs only in annelids, including DLamide, SLRFamide and QERAS (Fig. 1A). In addition, five pNPs did not have recognizable orthologs outside Urechis, and were temporarily classified as neuropeptides unique to U. unicinctus, including the FxFamide, FILamide, FW, FRWamide and ASYY (Fig. 1A).
Traditionally Echiura was ranked as a phylum, but recent studies, especially on molecular phylogenetic analysis [32] and morphological observation [33, 34], have generated an increasing body of evidence that they actually are derived annelids and provide strong support for a sister group relationship between Echiura and Capitellidae. This is consistent with our study in which we find three Annelid-specific pNPs were presented in U. unicinctus.
Screen of the neuropeptide precursors potentially involving in the larval settlement
We performed a hierarchical clustering of the neuropeptide precursors based on their stage-specific expression (FPKM values) from the U. unicinctus transcriptomes (Fig. 1B and Supplementary Fig. S2). The results showed that most of the neuropeptide precursors were expressed at multiple stages, and the expression levels were significantly different. We found when the larvae developed from LT to ES, a process which the larvae initially transited from upper to middle layer in the water column, expression levels of NPF-3 and DH44 decreased significantly (p < 0.05), while that of bursicon-A2 and NPF-1 was significantly increased (p < 0.05) (Fig. 1B and Supplementary Fig. S2). During the development progress from ES to SL, a period that the larvae move from the middle to the bottom of water layer, eleven pNP genes (MIP, bursicon-A2, NPF-2, RGWamide, 7B2, pedal peptide 1, myomodulin, FVRIamide, FxFamide, FILamide and FRWamide) were significantly up-regulated (p < 0.05) (Fig. 1B and Supplementary Fig. S2). However, eight genes (except 7B2, pedal peptide 1 and FVRIamide) among the eleven pNPs above were again down-regulated (p < 0.05) when the larvae developed from SL to WL, which is the period that the larvae begin to explore the suitable substrate and finally became benthic larvae (Fig. 1B and Supplementary Fig. S2). As MIP have been confirmed to regulate larvae settlement behavior [9], we speculated preliminarily the eight pNPs with similar expression pattern were considered to be most likely pNPs involved in the regulation of larval settlement and metamorphosis in U. unicinctus.
Sequence characteristics of the selected pNPs that may be involved in the regulation of larval settlement in U. unicinctus
Four interesting pNPs, including previously reported MIP [9] and three Uu-specific pNPs (FxFamide, FILamide and FRWamide), were selected for further analysis.
MIPs (Myoinhibitory peptides) are pleiotropic neuropeptides first described in insects as inhibitors of muscle contractions [18, 35, 36]. In some insect species, MIPs modulate juvenile hormone synthesis and reduce food intake, and they are also referred to as allatostatin-B or WWamide [37-40]. In Platynereis the MIPs have been confirmed to regulate larvae settlement behavior [9] and feeding behavior [41]. They are characterized by a conserved domain containing two Trp residues which are usually separated by five to eight amino acid residues in insects, molluscs and annelids [42-44]. In U. unicinctus transcriptomes, we identified a neuropeptide precursor which is an orthologue of arthropod MIP (Fig. 2). The Uu-MIP precursor contains 11 mature peptides, the number of mature peptides in U. unicinctus is the same as that of the annelid Platynereis dumerilii, while differs from the mollusc Patinopecten yessoensis and the arthropod Megabalanus volcano which have 10 mature peptides (Fig. 2A). Sequence alignment of the bioactive peptides revealed that the sequence similarity among the different mature MIPs in U. unicinctus was higher than that in P. dumerilii, P. yessoensis and M. volcano (Fig. 2B). Moreover, the MRVWamide motif in C-terminal of the mature MIPs is present in U. unicinctus and P. dumerilii, but not in P. yessoensis and M. volcano (Fig. 2B). The above results show that the characteristics of the MIP precursor sequence of U. unicinctus are closer to that of P. dumerilii, which is consistent with the classic species evolution.
In this study, five potential neuropeptide precursors were for the first time identified in U. unicinctus (Fig. 1A), and three of them (FRWamide, FILamide and FxFamide) were predicted to play a role in regulating U. unicinctus larvae settlement based on the significant differences in mRNA level from the segmented larvae to worm-shaped larvae (Fig. 1B). FRWamide precursor is comprised of 202 amino acids which contains a 25-residue signal peptide and 7 copies of neuropeptides with FRWamide motif in the C-terminal (Fig. 3A and D). FILamide precursor is comprised of 336 amino acids which contains a 27-residue signal peptide and 8 copies of neuropeptides with FILamide motif in the C-terminal (Fig. 3B and E). FxFamide precursor is comprised of 509 amino acids which contains a 33-residue signal peptide and 15 copies of neuropeptides with FxFamide motif in the C-terminal (Fig. 3C and F). These newly discovered neuropeptide precursors enrich the intension of neuropeptide composition.
Spatio-temporal expression of the selected pNPs during the larval settlement
To verify the expression of the four pNP transcripts (MIP, FILamide, FxFamide and FRWamide), U. unicinctus larvae including late-trochophore (LT), pre-competent larva (PL), competent larva (CL), post-competent larva (POL) and worm-shaped larva (WL) were employed for qRT-PCR analysis (Fig. 4A). The results showed that the mRNA levels of the four pNP genes increased through larval development, with the highest expression in CL, and then significant decrease in POL and WL (Fig. 4B). These results are consistent with the transcriptome data (Fig. 1B and Supplementary Fig. S2). During the developmental progression from LT to CL, the U. unicinctus larvae move from the upper to the middle layer in water, and gradually acquire the ability to explore a suitable substrate in CL, finally become benthic larvae in WL. Thus, we suggested that these four genes may be involved in the biological activities of the larvae exploring the substrate for settlement in U. unicinctus.
To map the expression of these pNPs (MIP, FxFamide, FILamide and FRWamide) to nerve cells at the special sites, nervous system in U. unicinctus larvae was analyzed using fluorescence immunohistochemistry with an anti-5HT antibody (Fig. 5). The results showed that, in trochophore up to an age of approximately 15 days, only a few structures of the nervous system are labeled with antibodies against 5-HT. In the episphere of the larvae, the circumoesophageal connectives (CC) and two nerve rings innervating the prototroch and metatroch are visible (Fig. 5A). In the hyposphere of the larva, two longitudinal nerves (LN) merge after a short distance forming a median nerve named ventral nerve cord (VNC). Two pairs of perikarya are discernible in the anterior region, directly behind the slit-shaped mouth opening (Fig. 5A, C) and on the telotroch nerve ring (Fig. 5A, B, C). In dorsal view of the larva, 3-4 LNs can be seen in the episphere which connect to the prototroch and metatroch nerve rings (Fig. 5B). As development proceeds up to the competent larva, in which the anterior chaetae have already been formed, the paired longitudinal nerve tracts of the VNC are fused in the ventral midline (Fig. 5C). In addition, the metatroch nerve ring is disappeared and two labeled perikaryas are visible on the dorsal side of the larva just under the prototroch nerve ring (Fig. 5D, E). The apical organ of the larva is shown in Fig. 5F. Fluorescence immunohistochemistry were also used to study the development of the nervous system in various other Echiuran species, such as Bonellia viridis [45, 46] and Urechis caupo [33]. Our results are consistent with those previous studies which have proven to be informative in the study of neurogenesis in neuronal structures of Echiurans. Besides, we revealed several previously unreported details — eight nerve fibers and six large labeled perikaryas are visible in the apical organ in Echiuran worm (Fig. 5F), which is similar to that reported in P. dumerilii [9, 21, 29, 47] and especially in Capitella teleta [47].
Next, location of four pNP mRNAs including MIP, FxFamide, FILamide and FRWamide were detected by Whole-mount mRNA in situ hybridization (WISH) (Fig. 6 and Fig. S3). The results showed that a positive MIP signal was first observed in the central region of the episphere in the early-trochophore larva (Fig. 6A) which is similar to that of the apical organ in C. teleta and P. dumerilii [9, 47]. As the development proceeds, four positive cells are exclusively located in the apical organ of the late-trochophore larva (Fig. 6B). Until the competent larvae, the obvious positive signals were located in four regions, including the apical organ (the 4-6 cells), above the abdomen chaetae (the two cells), the prototroch in the dorsal side of the larvae (the two cells) and the both side of the telotroch (the two cells) (Fig. 6C). The expression patterns of FRWamide, FxFamide and FILamide were similar to that of MIP (Fig. 6), except no visible FRWamide positive signal was observed in U. unicinctus early-trochophore (Fig. 6D), in competent larvae FxFamide was detected only in apical organ (the six stained cells) and above the abdomen chaetae (the two stained cells) (Fig. 6I), while the positive signal of FILamide in the competent larva was only in the four positive cells of apical organ (Fig. 6L). Since the WISH experiment was observed after being sealed with resin, it was difficult to observe the apical view of the larva.
In many marine invertebrates the transition from free-swimming larvae to bottom-dwelling juveniles is regulated by neuroendocrine signals (including neuropeptides and hormones) [48-50]. In diverse ciliated marine larvae, the apical organ, has been implicated in the detection of cues for the initiation of larval settlement [9, 10, 51-54]. Previous studies suggest that several neuropeptides expressed in distinct sensory neurons (apical organ) innervate locomotor cilia, which contribute to the larvae swimming depth [9, 21]. The alternation of active upward swimming and passive sinking, together with swimming speed and sinking rate, is thought to determine vertical distribution in the water [55]. In Platynereis, neuropeptides including RYa, FVMa, DLa, FMRFa, FVa, LYa, YFa, FLa, MIP, GWa et al can alter ciliary beat frequency and the rate of calcium-evoked ciliary arrests [9, 21], which eventually may be involved in regulating the larvae settlement. In our study, the MIP and FRWamide were detected in the dorsal side of the prototroch nerve ring and the both side of the telotroch nerve ring (Fig. 6C, F), indicating that they may play a role in U. unicinctus ciliary beating and eventually cause the larvae sinking to the bottom. MIP is the only neuropeptide that has been shown to be involved in larval settlement in Platynereis [9], which is expressed in chemosensory–neurosecretory cells in the annelid larval apical organ. The researchers found that synthetic MIPs can induce the settlement of P. dumerilii larvae, and they demonstrate by morpholino-mediated knockdown that MIP signals via a G protein-coupled receptor to trigger settlement [9]. These results reveal a role for a conserved MIP receptor–ligand pair in regulating marine annelid settlement. In this study, we revealed that MIP, FxFamide, FILamide and FRWamide all localized in the region of the apical organ (Fig. 6), like MIPs expression pattern in P. dumerilii, indicating that these neuropeptides may also be involved in triggering larval settlement. In addition, the expressions of the MIP, FRWamide and FxFamide were also detected at base of the abdomen chaetae (Fig. 6C, F and I). The chaetae are important in locomotion, stabilization during peristalsis, and sensing the environment in annelids [56], and have been implicated in assisting movement and stabilizing body segments within the tube for worms living in burrows or tubes [57, 58]. However, in sediment-dwellers they contact the inside walls of tubes or burrows. The cantilever nature of capillary chaetae and their astounding breadth of flexural stiffness suggest that they could be very effective at transmitting specific mechanical information about their surroundings to the body of the worm [33, 56]. Therefore, we propose that MIP, FRWamide and FxFamide located in base of the abdomen chaetae may help U. unicinctus larvae crawl on the sediment surface or explore the bottom and eventually contribute to larval settlement. Meanwhile, there are also some limitations to this study which need to be acknowledged. Firstly, this study only uses transcriptomic data to identify neuropeptides, therefore these predicted pNPs have not been confirmed by mass spectrometry to show that they are definitely released as signaling peptides in the worm. Secondly, we only used WISH technology to explore the location of the pNP genes at the mRNA level. Therefore, some issues including mass spectrometry, immunohistochemistry, western blot and other functional studies remained to be investigated in the future.