1. Introduction

The peptide calcitonin (CT) was first identified as a hormone that regulates blood calcium levels and subsequently a family of CT-related neuropeptides was discovered in mammals, including calcitonin gene related peptides (CGRPs), amylin (AMY), adrenomedullin (AM), and CT receptor-stimulating peptide (CRSP) (Copp et al., 1962; Sekiguchi et al., 2009; Sekiguchi et al., 2016). Members of the CT-type family of peptides typically have a disulfide bond in their N-terminal region (Cai et al., 2018) and exert their physiological effects in mammals by binding to two related G protein-coupled receptors: CTR (CT receptor) and CLR (CTR-like receptor) (Martins et al., 2014). However, receptor activity-modifying proteins (RAMPs) acting as accessory proteins are key determinants of ligand selectivity for CTR and CLR (Mclatchie et al., 1998). Thus, formation of a complex with a RAMP is essential for CLR function, whereas RAMPs are not required for CTR ligand recognition (Kuwasako et al., 2011; Bomberger et al., 2012). Furthermore, binding of CT to CTR in the absence of RAMPs (Katafuchi et al., 2009) can activate signaling via several downstream pathways, including cAMP accumulation, Ca²⁺ mobilization, and ERK activation (Chen et al., 1998; Pondel, 2000; Walker et al., 2010; Andreassen et al., 2014).

Transcriptome/genome sequencing has enabled analysis of the phylogenetic distribution of CT-type signaling systems, indicating that the evolutionary origin of CT-type peptides and CTR/CLR-related receptors can be traced back to the urbilaterian common ancestor of deuterostomes and protostomes (Mirabeau and Joly, 2013; Jekely, 2013). Furthermore, in protostomes two different types of CT-related peptides have been identified. Firstly, peptides that are structurally similar to vertebrate CT-type peptides in having an N-terminal disulphide bridge and secondly, diuretic hormone 31 (DH₃₁)-type peptides that lack an N-terminal disulphide bridge. Furthermore, phylogenetic analysis indicates that gene duplication in a common ancestor of the protostomes gave rise to genes encoding CT-like peptides with a disulphide bridge and DH₃₁-type peptides, which were then differentially retained in protostome taxa (Cai et al., 2018). Receptors for CT-like peptides with a disulphide bridge have been characterised in a molluscan species, the oyster Crassostrea gigas, which has seven CTR/CLR-type receptors. Two of these receptors, named Cg-CT-R and Cragi-CTR2, were shown to be specifically activated by the CT-type neuropeptides Cragi-CT1b and Cragi-CT2, respectively, thereby mediating intracellular Ca²⁺ mobilization and cAMP accumulation (Schwartz et al., 2019). CT-like peptides with a disulphide bridge are not present in the insect Drosophila melanogaster. However, receptors that are activated by DH₃₁ have been identified in this species and, interestingly, DH₃₁ can act as a ligand for two types of receptors. Thus, DH₃₁ acts as a ligand for a CTR/CLR-related protein (CG17415), but it also acts as a ligand for a protein (CG13758) that is the receptor for pigment-dispersing factor (PDF) (Mertens et al., 2005; Shafer et al., 2008). However, the evolutionary and functional significance of the occurrence of dual receptor pathways for DH₃₁ in Drosophila are not known.

Currently, relatively little is known about CT-type signaling systems in deuterostome invertebrates. Analysis of transcriptome/genome sequence data has enabled identification of genes/transcripts encoding CT-type peptides and candidate CTR/CLR-type receptors for these peptides in several deuterostome invertebrate taxa, including urochordates (e.g. Ciona intestinalis), cephalochordates (e.g. Branchiostoma floridae), hemichordates (e.g. Saccoglossus kowalevskii) and echinoderms (e.g. Strongylocentrotus purpuratus) (Burke et al., 2006; Sekiguchi et al., 2009; Sekiguchi et al., 2016; Rowe and Elphick, 2012; Martins et al., 2014). Furthermore, analysis of the expression and action of CT-type peptides identified in the starfish Asterias rubens and Patiria pectinifera has revealed that they act as muscle relaxants (Cai et al., 2018; Semmens et al., 2016). However, experimental confirmation of ligand-receptor partners has thus far only been reported for the cephalochordate B. floridae, where three CT-type peptides were shown to act as ligands for a CTR/CLR-type receptor when it is co-expressed with RAMP-type proteins (Sekiguchi et al., 2016). To gain further insights into the mechanisms of CT-type signaling in invertebrate deuterostomes, the aim of this study was to functionally characterise CT-type peptides and their receptors in an echinoderm – the sea cucumber Apostichopus japonicus.

• A. japonicus is an economically important species in Asian aquaculture (Pangestuti and Arifin, 2018; Guo et al., 2020) and a species representative of the phylum Echinodermata. As a phylum of deuterostome invertebrates, echinoderms form a sister clade to the chordates together with hemichordates and xenoturbellids (Bromham and Degnan, 1999; Bourlat et al., 2006; Dunn et al., 2008) and as such they occupy a key “intermediate” phylogenetic position relative to the protostome invertebrates and vertebrates (Zandawala et al., 2017), which make them attractive as experimental systems to address evolutionary questions. In our previous studies, we have established this species as a model system to obtain insights into the evolutionary history and comparative physiology of neuropeptide signaling systems in bilaterians (Chen et al., 2019; Li et al., 2022; Zheng et al., 2022; Cong et al., 2023; Zheng et al., 2024). A gene encoding two CT-type peptides (AjCT1 and AjCT2) has been identified in A. japonicus (Chen et al., 2019) and a candidate receptor (AjCTR) for these peptides has also been predicted (Huang et al., 2021). Furthermore, relevant to the discovery that the CT-like peptide DH₃₁ can act as a ligand for both a CTR/CLR-type receptor and a PDF-type receptor in Drosophila, the sequences of two PDF-type receptors have been identified in A. japonicus (AjPDFR1 and AjPDFR2) (Huang et al., 2021).

The first aim of this study was to investigate if AjCT1 and/or AjCT2 act as ligands for AjCTR, AjPDFR1 and AjPDFR2 and to characterise the downstream signaling pathways that are activated when AjCT1 and/or AjCT2 bind to their receptor(s). The second aim was to investigate the physiological roles of AjCT1 and AjCT2 in A. japonicus by analysing the expression of the gene encoding these peptides, examining the in vitro and in vivo pharmacological effects of these peptides and performing loss-of-function tests. The functional characterisation of CT-type signaling in A. japonicus reported in this study provides new insights into the evolution and comparative physiology of neuropeptides and a basis for application of neuropeptide-based strategies to improve aquaculture methods for this economically important species.

2. Results

2.1 Characterization and phylogenetic analysis of AjCT1/2

The complete cDNA sequences of the CT-type neuropeptide precursors AjCTP1 and AjCTP2 were confirmed. The coding sequence of AjCTP2 is shorter than AjCTP1 (Figure 1A) but both transcripts encode precursor proteins that comprise the same predicted 23-residue N-terminal signal peptide (Figure 1-figure supplement 1A). AjCTP1 encodes both putative CT-type neuropeptides (AjCT1 and AjCT2), while AjCTP2 only encodes AjCT2 (Figure 1A, Figure 1-figure supplement 1A and 1B). Exon and intron structure analysis showed that AjCTP1 comprises four exons, with AjCT1 and AjCT2 encoded in the third and fourth exons, respectively. AjCTP2 comprises three exons, excluding the third exon that encodes AjCT1 (Figure 1A). Phylogenetic analysis revealed that AjCTP1 and AjCTP2 are closely related to CT-type precursors from other sea cucumbers (Holothuria glaberrima and Holothuria scabra), and are positioned within a clade that also contains CT-type precursors from other echinoderms (sea urchin S. purpuratus, starfish A. rubens). Furthermore, with vasopressin/oxytocin-type neuropeptide precursors included as an outgroup, the echinoderm CT-type precursors are positioned within a clade that also comprises CT-type precursors from chordates and DH₃₁/CT-type precursors from protostomes (Figure 1B). The predicted tertiary structures of the AjCTP1 and AjCTP2 proteins are shown in Figure 1-figure supplement 1C. Sequence alignment of CT-related neuropeptides revealed that, with the exception of DH₃₁-type peptides, the N-terminal region of CT-related peptides has a pair of conserved cysteine residues that are known or predicted to form a disulfide bond. Another conserved feature is a C-terminal proline residue, with the exception of CGRPs (Figure 1-figure supplement 2). Accordingly, both AjCT1 and AjCT2 have these two conserved characteristics described above.

Figure 1.

2.2 Characterization and phylogenetic analysis of three candidate receptors for AjCT1/2

The sequences of three candidate receptors for AjCT1 and AjCT2 were obtained by RACE and phosphorylation sites were predicted, as shown in Figure 1-figure supplement 3A, 3B, and 3C. Phylogenetic analysis revealed that CTR/CLR-type receptors divided into two clades and AjCTR was positioned in a clade with other deuterostome CTR/CLR-type receptors, whilst the other clade comprised CTR/CLR-type receptors from protostomes. Similarly, AjPDFR1 and AjPDFR2 were positioned in a clade comprising PDF-type receptors from other deuterostomes, whilst another clade comprised PDF-type receptors from protostomes (Figure 1C). Domain prediction of the candidate receptors showed that AjCTR had HRM (Hormone receptor) and 7tm_GPCRs (seven-transmembrane G protein-coupled receptor) superfamily domains (Figure 1-figure supplement 4A), whereas AjPDFR1 and AjPDFR2 only presented the 7tm_GPCRs superfamily domain (Figure 1-figure supplement 4B). To further investigate an orthologous relationship of the candidate receptors with homologous receptors in other echinoderms, we conducted a gene synteny analysis in A. japonicus, A. rubens and L. variegatus. The neighboring genes of AjCTR included GOLGA4, NeuroD, CRF2, ppGalNAc-T, URB3, SLC40A1, CMAH and MAGP. The relative positions of these genes were different from those in A. rubens and L. variegatus (Figure 1-figure supplement 4C). The same situation was also observed for the synteny analysis of PDFRs.

2.3 AjCT1 and AjCT2 act as ligands for AjCTR, AjPDFR1 and AjPDFR2

To investigate if AjCT1 and/or AjCT2 act as ligands for AjCTR, AjPDFR1 and AjPDFR2, here, two receptor assay methods were employed. As shown in Figure 2A, both AjCT1 and AjCT2 (10^-6 M) caused a significant increase in CRE luciferase activity in HEK293T cells transfected with AjCTR, AjPDFR1 or AjPDFR2 (Table 1). Furthermore, this effect was inhibited by pre-incubation of cells with the PKA inhibitor H89. The intracellular accumulation of cAMP was also measured, and this showed that AjCT1 and AjCT2 (10^-9 – 10^-5 M) caused a dose-dependent increase in cAMP in HEK293T cells transfected with AjCTR, AjPDFR1 or AjPDFR2 (Figure 2B). However, when exposed to AjCT1 or AjCT2, the maximum accumulation of intracellular cAMP was higher in cells transfected with AjCTR than in cells transfected with AjPDFR1 and AjPDFR2 (Figure 2B). Measurement of SRE luciferase activity revealed that AjCT1 and AjCT2 (10^-6 M) administration evoked an increase in activity in HEK293T cells transfected with AjCTR and AjPDFR1 (Figure 3A). However, in HEK293T cells transfected with AjPDFR2, an increase in SRE luciferase activity was only observed with AjCT2, not with AjCT1 (Table 1). Furthermore, as shown in Figure 3A, AjCT1- and AjCT2-induced increases in SRE luciferase activity were not observed in cells that had been pre-incubated with FR900359 (Gαq protein inhibitor) and Gö 6983 (PKC inhibitor). As shown in Figure 3-figure supplement 1, receptors localized in the cell membrane, but after exposure to AjCT1 and AjCT2 (10^-6 M) for 15 min, internalization of pEGFP-N1/AjCTR, pEGFP-N1/AjPDFR1 and pEGFP-N1/AjPDFR2 was observed in transfected HEK293T cells (Figure 3B). Taken together, all these findings indicate that AjCT1 and AjCT2 can act as ligands for the three candidate receptors and then activate cAMP/PKA signaling and Gαq/Ca²⁺/PKC signaling in all combinations, with the exception of AjCT1/AjPDFR2 which only activates the cAMP/PKA cascade.

Figure 2.

Figure 3.

Table 1.

To investigate whether the activated signaling pathways may exert downstream effects via the ERK–MAPK pathway, the phosphorylation levels of ERK1/2 were analysed in transfected HEK293T cells. pERK1/2 levels in cells transfected with AjCTR or AjPDFR1, but not AjPDFR2, were significantly increased after a 5 min incubation with AjCT1 (Figure 4A). Furthermore, this effect was blocked by the PKC inhibitor in AjCTR-expressing cells, and this effect was blocked by both PKA and PKC inhibitors in AjPDFR1-expressing cells (Figure 4-figure supplement 1A). pERK1/2 levels in cells transfected with AjCTR or AjPDFR1 or AjPDFR2, were significantly increased after a 5 min incubation with AjCT2 (Figure 4B). Moreover, this effect persisted until 15 min after exposure to AjCT in both AjCTR-transfected cells and AjPDFR1-transfected cells but not in AjPDFR2-transfected cells (Figure 4B, Table 1). Furthermore, these effects were blocked by PKA and PKC inhibitors in AjPDFR1 and AjPDFR2 expressing cells, whereas in AjCTR expressing cells, peptide-induced pERK1/2 activity was only blocked by a PKC inhibitor (Figure 4-figure supplement 1B). These results indicate that, with the exception of AjCT1/AjPDFR2, both AjCT1 and AjCT2 can activate the ERK1/2 cascade by binding to the candidate receptors.

Figure 4.

2.4 Localization of AjCTP1/2 expression in A. japonicus

qRT-PCR analysis revealed that AjCTP1/2 had the highest expression level in the circumoral nervous system (CNS) compared with other tissues (p < 0.05) (Figure 5A).

Figure 5.

Informed by these findings, mRNA in situ hybridization was used to visualize transcripts in the CNS and the AjCTP1/2 positive signals were observed (Figure 5B). No positive staining was detected in the CNS incubated with the sense probe (Figure 5-figure supplement 1).

2.5 AjCT1 and AjCT2 causes dose-dependent relaxation of longitudinal muscle and intestine preparations from A. japonicus

Both AjCT1 and AjCT2 caused dose-dependent relaxation of longitudinal muscles from A. japonicus at 10^-10–10^-6 M. At the highest concentration tested (10^-6 M), AjCT1 and AjCT2 caused 30.07 ± 7.49% and 32.50 ± 14.49% reversal of ACh-induced contraction, respectively (Figure 6A, 6B). AjCT1 had no effect on the contractile state of intestine preparations, even at concentrations as high as 10^-5 M (Figure 6C). Unlike AjCT1, AjCT2 caused dose-dependent relaxation of intestine preparations, with 26.33 ± 8.75% reversal of ACh-induced contraction observed at 10⁻⁵ M (Figure 6D).

Figure 6.

2.6 AjCT2 affects feeding and growth in A. japonicus

To investigate the effects of AjCT1 and AjCT2 in vivo, a long-term injection experiment was carried out. As shown in Figure 7A, there were no significant changes in wet mass in the CT1L, CT1H and CT2L groups (p ≥ 0.05), but in the CT2H group there was a significant increase in wet weight after 24 days of injection in comparison with the control group (CO) (p < 0.05). Correspondingly, the mass of faeces increased and the residual bait mass decreased (Figure 7A-figure supplement 1). Furthermore, the relative intestinal expression levels of several growth factors were investigated and the results showed that AjGDF-8, a growth inhibitory factor, was significantly down-regulated in all the experimental groups. In contrast, the relative expression levels of AjIgf and AjMegf6, growth promoting factors, were significantly up-regulated in intestines (Figure 7B).

Figure 7.

Subsequently, the relative intestinal expression levels of AjCT1 and AjCT2 receptors were further tested for the CT2H group. The results showed that the relative expression levels of all three receptors were significantly up-regulated, with the ranked fold change being AjCTR > AjPDFR1 > AjPDFR2 (Figure 7C). Conversely, the relative expression levels of the three receptor genes were significantly down-regulated in siAjCTP1-1 treated animals (Figure 8A, 8B and 8C). The interference rate for siAjCTP1/2-1 was 66 % (Figure 8-figure supplement 1).

Figure 8.

3. Discussion

Previous studies have revealed that the CT-type neuropeptide DH₃₁ can act as a ligand for a CTR/CLR-type receptor and a pigment-dispersing factor (PDF) receptor in a protostome – the insect D. melanogaster (Mertens et al., 2005; Shafer et al., 2008; Schwartz et al., 2019). Here we show that two CT-type peptides (AjCT1 and AjCT2) act as ligands for a CTR/CLR-type receptor and two pigment-dispersing factor (PDF) type receptors in a deuterostome – the sea cucumber A. japonicus (phylum Echinodermata). This is an important finding because it indicates that the ability of CT-type neuropeptides to act as ligands for both CTR/CLR-type receptors and PDF-type receptors is an evolutionarily ancient property that originated in a common ancestor of the Bilateria. However, further studies on a wider variety of both protostome (e.g. molluscs, annelids) and deuterostome taxa (e.g. other echinoderms, hemichordates) will be needed to establish if this is an evolutionarily conserved property of CT-type neuropeptides. Here, we also investigated the signal transduction pathways that are activated by AjCT1 and AjCT2 when these peptides bind to their receptors and then proceeded to investigate the expression and actions of these peptides to gain insights into their physiological/behavioral roles in A. japonicus, as discussed below.

3.1 Alternative RNA splicing gives rise to transcripts encoding one (AjCT2) or two (AjCT1, AjCT2) CT-type neuropeptides in A. japonicus

In vertebrates, alternative RNA splicing gives rise to transcripts encoding CT or CGRP (Amara et al., 1982; Morris et al., 1984). Analysis of transcriptome/genome sequence data from A. japonicus has revealed that in this species alternative RNA splicing gives rise to transcripts encoding one (AjCT2) or two (AjCT1, AjCT2) CT-type neuropeptides (Rowe et al., 2014; Zandawala et al., 2017; Chen et al., 2019; this study). A similar pattern of alternative RNA splicing has been reported for CT-type precursors in other sea cucumbers (H. scabra and H. glaberrima; class Holothuroidea) and in brittle stars (Amphiura filiformis and Ophiopsila Aranea; class Ophiuroidea) (Zandawala et al., 2017; Suwansa-Ard et al., 2018). In contrast, CT-type precursors identified in other echinoderm classes (Asteroidea, Echinoidea and Crinoidea) comprise a single CT-type neuropeptide (Rowe and Elphick, 2012; Mirabeau and Joly, 2013; Rowe et al., 2014; Semmens et al., 2016; Cai et al., 2018; Aleotti et al., 2022). However, it remains to be determined if this feature of CT-type precursors in sea cucumbers and brittle stars evolved independently or originated in a common ancestor of eleutherozoan echinoderms but with subsequent loss in Asteroidea and Echinoidea (Figure 9).

Figure 9.

Analysis of the sequences of AjCT1 and AjCT2 revealed the presence of a two cysteine residues in their N-terminal regions, which is an evolutionarily conserved feature of CT-type peptides. Furthermore, structural analysis of CT-type peptides in vertebrates and in starfish has revealed that these cysteines form a disulphide bridge in the mature peptides (Cai et al., 2018) and therefore it is inferred that this post-translational modification also occurs in AjCT1 and AjCT2. Furthermore, the C-terminal residue of the AjCT1 and ACT2 is predicted to be an amidated proline, which is a conserved post-translational modification of CT/DH31-type peptides in many taxa, but not vertebrate CGRPs (Cai et al., 2018).

3.2 Identification and characterisation of receptors for CT-type neuropeptides in A. japonicus

Informed by previous genome-wide analysis of GPCRs in A. japonicus (Huang et al., 2021), we identified three candidate receptors for AjCT1 and/or AjCT2. Firstly, AjCTR, which is a homolog of CT/DH₃₁-type receptors that have been identified in other taxa, including vertebrates and insects (Garelja et al., 2022; Johnson et al., 2005; Toribio et al., 2003). Secondly, AjPDFR1 and AjPDF2, which are homologs of the PDF-type receptor that has been shown to be activated by DH₃₁ in D. melanogaster (Mertens et al., 2005; Zandawala, 2012).

In mammals, CTR and CLR mediate effects of all CT-related neuropeptides, but their ligand specificity relies on the presence and types of receptor activity-modifying proteins (RAMPs) (McLatchie et al., 1998; Purdue et al., 2002; Conner et al., 2004; Hay et al., 2018). Thus, CTR acts as a functional receptor for CT-type peptides without an associated RAMP (Katafuchi et al., 2009), whereas activation of downstream signaling via CLR requires an associated RAMP (Hay et al., 2018). Here, we investigated the occurrence of RAMPs in A. japonicus using the tblastn method employed by Sekiguchi et al., (2016), which enabled identification of RAMPs in the cephalochordate B. floridae. However, no RAMPs were identified in A. japonicus, suggesting that AjCTR may be functionally more similar to CTR than CLR. Accordingly, tblastn analysis of other available echinoderm genomes (A. rubens, S. purpuratus and A. japonica), as well as transcriptome databases for brittle stars (O. victoriae, O. aranea and A. filiformis; no published genomes), also failed to identify RAMPs. Likewise, RAMPs have also not been found in protostomes (Schwartz et al., 2019). Therefore, RAMPs may be unique to chordates, although interestingly RAMPs have also not been identified in the urochordate C. intestinalis (Sekiguchi et al., 2009).

As highlighted above, here we show that the CT-type peptides AjCT1 and AjCT2 both act as ligands for a CTR/CLR-type receptor (AjCTR) and two PDF-type receptors (AjPDFR1, AjPDFR2) in A. japonicus. Furthermore, characterization of downstream signaling mechanisms revealed that both AjCT1 and AjCT2 trigger AjCTR-mediated cAMP accumulation, Ca²⁺ mobilization, and ERK signaling. These findings are consistent with previous studies on CT-type receptors in vertebrates and in the mollusc Crassostrea gigas (Pondel, 2000; Walker et al., 2010; Andreassen et al., 2014; Schwartz et al., 2019). Both AjCT1 and AjCT2 trigger cAMP accumulation, Ca²⁺ mobilization, and ERK signaling mediated by AjPDFR1. Both AjCT1 and AjCT2 triggered cAMP accumulation mediated by AjPDF2, but only AjCT2 (not AjCT1) triggered Ca²⁺ mobilization and ERK signaling mediated by AjPDFR2. These differences in the signal transduction mechanisms activated by AjCT1 and AjCT2 may account for differences in the pharmacological activities and physiological roles of these peptides, as discussed below.

3.3 AjCT1 and AjCT2 act as muscle relaxants in A. japonicus

Analysis of the expression of the gene encoding the precursors comprising AjCT2 or AjCT1 and AjCT2 revealed the occurrence of transcripts in the circumoral nervous system as well as in peripheral tissues/organs, including the longitudinal body wall muscle, digestive system and gonads. Accordingly, analysis of CT-type neuropeptide expression in other echinoderms has revealed a widespread pattern of expression in larval and adult starfish (A. rubens) and in juvenile and adult featherstars (Antedon mediterranea) (Mayorova et al., 2016; Cai et al., 2018; Aleotti et al., 2022). Several CT-related peptides act as muscle relaxants in vertebrates (Steiner et al., 1991; Pinto et al., 1996; Yoshimoto et al., 1998; Edvinsson et al., 2001; Golpon et al., 2001; Kandilci et al., 2008) and, accordingly, previous studies have revealed that CT-type neuropeptides cause relaxation of muscle preparations in starfish, including locomotory organs (tube feet) and the apical muscle, which is functionally similar to the longitudinal body wall muscle in sea cucumbers (Cai et al., 2018). Therefore, it was of interest to investigate if AjCT1 and/or AjCT2 act as muscle relaxants in A. japonicus. Both AjCT1 and AjCT2 caused dose-dependent relaxation of longitudinal muscle preparations, consistent with the relaxing effect of CT-type peptides on starfish apical muscle preparations. Furthermore, AjCT2, but not AjCT1, caused dose-dependent relaxation of intestine preparations from A. japonicus. Interestingly, the CT-type peptide ArCT does not to cause relaxation of cardiac stomach preparations from the starfish A. rubens (Cai et al., 2018). Therefore, from a functional perspective ArCT is more like AjCT1 than AjCT2 and we speculate that this may reflect loss of an exon encoding a AjCT2-like peptide in the Asteroidea lineage. Further insights into this issue and the evolution of CT-type neuropeptide function echinoderms could be obtained by investigation of the actions of CT-type peptides in brittle stars and sea urchins. Differences in the effects of AjCT1 and ArCT2 on the intestine may be explained by differences in their signal transduction mechanisms. Thus, only AjCT2, not AjCT1, triggered AjPDFR2-mediated acivation of Ca²⁺ mobilization and ERK signaling and therefore we speculate that the relaxing effect of AjCT2 on the intestine may be mediated by activation of AjPDFR2.

3.4 AjCT2 stimulates feeding and growth in A. japonicus

In vivo pharmacological experiments revealed that treatment with AjCT2 at a high concentration (5 × 10^-1 mg/ml) caused a significant increase in body mass, and this was accompanied by a decrease in residual bait and an increase in feacal mass. However, these effects were not observed with AjCT2 at a low concentration (5 × 10^-3 mg/ml) or with AjCT1 at low or high concentrations. It is noteworthy that AjCT2, but not AjCT1, has a relaxing effect on the intestine in A. japonicus and therefore the effects of AjCT2 on feeding, defecation and growth may, at least in part, be mediated via binding of this peptide to receptors in the intestine. Accordingly, upregulation of the expression of the AjCT1/AjCT2 receptors AjCTR, AjPDFR1 and AjPDFR2 was observed in the intestine of animals injected with AjCT2 at a high concentration. Furthermore, changes in intestinal expression of genes encoding growth-related factors were also observed in animals injected with AjCT2 at a high concentration, with up-regulation of AjIgf and AjMegf6 expression and down-regulation of AjGDF-8 (Appendix 1—figure 1). Consistent with these changes in gene expression and the growth-promoting effect of AjCT2, it has been reported that GDF-8 inhibits growth in A. japonicus (Li et al., 2016), whilst Megf6 and Igf promote growth in vertebrates (Párrizas and LeRoith, 1997; Deribe et al., 2009).

Collectively, these findings indicate that AjCT2 has a physiological role in stimulating feeding and growth in A. japonicus. To the best of our knowledge, this is the first study to report orexigenic and growth-promoting effects of a CT-type neuropeptide in bilaterian animals. Interestingly, previous studies have reported the expression of CT-type neuropeptides in the digestive system of invertebrate chordates (Sekiguchi et al., 2009; Sekiguchi et al., 2016). However, in contrast to the stimulatory effect of AjCT2 on feeding/growth in A. japonicus, CT-type neuropeptides have been found to inhibit feeding and growth in vertebrates (Del et al., 2002; Hay et al., 2018; Nakamura et al., 2018). Thus, it appears that opposing roles of CT-type neuropeptide signaling systems as regulators for feeding/growth have evolved in deuterostomes, with a stimulatory role reported here in an echinoderm and an inhibitory role reported previously in vertebrates. Further research is now needed to investigate if the stimulatory effect of AjCT2 on feeding/growth observed in here in A. japonicus also applies to other sea cucumber species. Moreover, our discovery of this effect of AjCT2 on A. japonicus may provide a basis for development of novel approaches to improve aquaculture of this economically important species.

4. Materials and methods

4.1 Animals

Adult sea cucumbers were collected from an aquaculture breeding farm in Weihai, Shandong, China. All the animals were acclimated in breeding aquaria (18 ℃, 32 ppt) for at least seven days and fed daily with a commercially formulated diet. Tissues used for cDNA cloning and qRT-PCR were obtained from five adult A. japonicus (∼120 g) and kept at -80 ℃ until use. Circumoral nervous tissues from three adult sea cucumbers (∼120 g) were fixed with 4% paraformaldehyde (PFA, G-CLONE, Beijing, China) and used for in situ hybridization analysis. In vitro functional analysis and in vivo experiments were carried out using twelve (∼120 g body weight) and 168 (∼20 g) sea cucumbers, respectively.

Ethical approval

All methods were carried out in accordance with the ARRIVE guidelines (https://arriveguidelines.org) for animal experiments. All animal care and use procedures were approved by the Institutional Animal Care and Use Committee of Ocean University of China (Permit Number: 20141201). They were performed according to the Chinese Guidelines for the Care and Use of Laboratory Animals (GB/T 35892-2018).

4.2 Molecular structure and phylogenetic analysis of CT-type neuropeptides and precursors

A. japonicus CT-type neuropeptide precursor sequences (AjCTP1, GenBank accession number: MF401985; AjCTP2, GenBank accession number: MF401986) were identified in our previous study (Chen et al., 2019). Based on the published sequences, transcript-specific primers for AjCTP1 and AjCTP2 (Supplementary Table 1) were designed using Primer3Plus (https://www.primer3plus.com/) and sequence accuracy was confirmed by SMARTer RACE 5’/3’ Kit (TaKaRa, Kusatsu, Japan) according to the manufacturer’s instruction. A sequence translation tool (http://www.bio-soft.net/sms/index.html) was used to obtain the corresponding amino acid sequences encoded by the cloned cDNAs. To identify the conserved features of CT-type neuropeptides in Bilateria, sequences were aligned using Clustal-X with default settings and then predicted using the online tool Multiple Sequence Comparison Display Tool (http://www.bio-soft.net/sms/index.html). Tertiary structures were investigated using SWISS-MODEL (https://swissmodel.expasy.org/interactive). A neighbor joining tree was constructed to investigate relationships of AjCTP1, AjCTP2 and CT-type precursors from other Bilateria. Amino acid sequences were aligned using MEGA7 (v.7170509) with default settings and then modified by iTOL (https://itol.embl.de/login.cgi). The accession numbers or citations for all sequences used for analysis are listed in Supplementary Table 2.

4.3 Cloning, sequence analysis and phylogenetic tree construction for candidate AjCT1/2 receptors in A. japonicus

Five potential receptors for AjCT1 and AjCT2 in A. japonicus (GenBank accession number: PIK49998.1, PIK49999.1, PIK49604.1, PIK34007.1 and PIK62458.1) were identified based on genome-wide analysis (Huang et al., 2021). Sequence comparison revealed that both PIK49998.1 and PIK62458.1 were partial sequences of PIK34007.1. Thus, three candidate receptors named as AjCTR (Calcitonin receptor, GenBank accession number: PIK49604.1), AjPDFR1 (Pigment-dispersing factor receptor 1, GenBank accession number: PIK34007.1), and AjPDFR2 (Pigment-dispersing factor receptor 2, GenBank accession number: PIK49999.1) were predicted and then analyzed. To confirm the accuracy of these sequences, cDNAs were cloned using the SMARTer RACE 5’/3’ Kit (Takara, Cat No. 634858) and then sequenced (Beijing Genomics Institution, China). Receptor sequence analysis and phylogenetic analysis were performed as described in 4.2. In addition, the online tool NetPhos-3.1 (https://services.healthtech.dtu.dk/service.php?NetPhos-3.1) was used to predict potential phosphorylation sites. The conserved domains of protein sequences were predicted by the Batch SMART plugin of TBtools (Toolbox for biologists, v.1.098652) software. For analysis of gene synteny, neighboring genes of AjCTR, AjPDFR1 and AjPDFR2 were found based on an A. japonicus chromosome-level genome (Wang et al., 2022) and the corresponding genes in the starfish Asterias rubens and the sea urchin Lytechinus variegatus were also identified by performing BLASTp in the NCBI database. Specific primers for cloning of cDNAs encoding the candidate receptors are shown in Supplementary Table 1 and the details of the sequences used for constructing the phylogenetic tree are listed in Supplementary Table 3.

4.4 Testing AjCT1 and AjCT2 as ligands for candidate receptors and exploration of downstream signaling pathways

AjCT1 and AjCT2 were custom synthesized by GL Biochem Ltd. (Shanghai, China). Subsequently, CRE (cAMP response element) -Luc (Luciferase) detection, cAMP accumulation, SRE (serum response element) -Luc detection, receptor translocation assays, and Western blotting were carried out.

AjCTR, AjPDFR1 and AjPDFR2 incorporating enhanced green fluorescent protein (EGFP) were constructed respectively according to the method described by Li et al. (2022). Briefly, the complete ORFs of candidate receptors were cloned and sequenced. Then, the PCR products were digested with EcoRI and BamHI (TaKaRa, Kusatsu, Japan) and then incorporated into the pEGFP-N1 vector (YouBio, Changsha, China) that was subject to the same double-digestion. Finally, the constructed plasmid with correct sequences was transfected into HEK293T cells seeded on glass coverslips in 12-well plates. After incubation with AjCT1 (10^-6 M), AjCT2 (10^-6 M) or serum-free DMEM (Biosharp, Hefei, China) for 15 min, cells were washed with PBS (Biosharp, Hefei, China) and fixed with 4% PFA for 10 min. Finally, cell nuclei were stained with DAPI (G-CLONE, Beijing, China) for 5 min and an Olympus bright field light microscope was used for image capture. The pcDNA 3.1(+) vectors (YouBio, Changsha, China) comprising the complete ORF of the three candidate receptors were also constructed as described by Li et al. (2022). Then, they were co-transfected with the cAMP response element, pCRE-Luc (YouBio, Changsha, China) or the pSRE-Luc plasmid including PKC reporter gene (YouBio, Changsha, China) into HEK293T cells. Incubation was carried out with AjCT1 (10^-6 M), AjCT2 (10^-6 M) or serum-free DMEM (negative control) for 4 h at 37 °C. Finally, luciferase activity was tested using the firefly luciferase assay kit (MK, Shanghai, China). When required, transfected cells described above were co-incubated with H89 (PKA inhibitor) (Absin, Shanghai, China), FR900359 (Gαq protein inhibitor) (Cayman Chemical, Ann Arbor, Michigan) or Gö 6983 (PKC inhibitor) (Cayman Chemical, Ann Arbor, Michigan) before the experiment to explore the potential signaling pathways. To confirm the concentration of intracellular cAMP after stimulation, HEK293T cells expressing pcDNA 3.1(+)/AjCTR, pcDNA 3.1(+)/AjPDFR2 or pcDNA 3.1(+)/AjPDFR1 were treated with AjCT1 or AjCT2 (10^-9 – 10^-5 M) respectively for 15 min after 2 h starvation in serum-free medium. Subsequently, cAMP was measured using the ELISA-based cAMP assay kit (R&D systems, USA&Canada) following the manufacturer’s instruction. HEK293T cells transfected with empty pcDNA3.1(+) were used as a negative control. The specific primers used for constructing plasmids are listed in Supplemental Table 1.

Western blotting was performed as described previously (Li et al., 2022). Briefly, HEK293T cells transfected with the constructed pcDNA 3.1(+) plasmid were starved in serum-free medium for 2 h, and then incubated with AjCT1 or AjCT2 (10^-6 M). PVDF membranes containing proteins were blocked with 5% BSA (G-CLONE, Beijing, China) and incubated overnight with phosphorylated ERK1/2 kinases (CST, Boston, China). After that, goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Absin, Shanghai, China) was applied and incubated, followed by the addition of Omni-ECL™ Femto Light Chemiluminescence reagents (EpiZyme, Shanghai, China) . Finally, RVL-100-G (ECHO, America) was applied for visualization of the PVDF membrane. When required, transfected cells were co-incubated with PKA or PKC inhibitor before the experiment to identify signaling pathways that activate downstream ERK1/2 cascade.

4.5 Total RNA extraction and Quantitative Real-Time PCR (qRT-PCR) detection

qRT-PCR was employed to detect AjCTP1 and AjCTP2 transcript expression in five tissues from A. japonicus, including the intestine, longitudinal muscle, respiratory tree, gonad and circumoral nervous system. Because it was difficult to design specific primers for AjCTP1 and AjCTP2 (Zheng et al., 2022), total transcript expression levels for AjCTP1/2 (AjCTP1 + AjCTP2) were investigated. Total RNA was extracted from the collected tissues using Trizol RNA isolation reagent (Vazyme, Nanjing, China), cDNA was synthesized using Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix (YEASEN, Shanghai, China) according to manufacturer’s instructions. For qRT-PCR, amplification was performed in 20 µL volume reactions based on Hieff UNICON Universal Blue qPCR SYBR Green Master Mix (YEASEN, Shanghai, China) and expression levels were assessed by a Corbett Rotoe-Gnen Q (Qiagen, Germany). 𝛽 - actin (GenBank accession number: PIK61412.1) and 𝛽 -tubulin (GenBank accession number: PIK51093) were used as house-keeping genes for normalization (Zhao et al., 2014). The 2^−ΔΔCT method was applied to analyze the relative expression levels and comparison between tissues was carried out using one-way analysis of variance (ANOVA) followed by a Tukey post hoc test (IBM SPSS 25.0 software, Chicago, IL, USA). A p value < 0.05 was considered as a statistically significant difference. Specific sequences of primers used for qRT-PCR are listed in Supplemental Table 1.

4.6 Localization of AjCTP1/2 expression in A. japonicus using in situ hybridization

Digoxigenin-labeled RNA antisense probes and corresponding sense probes were synthesized using DIG RNA Labelling Kit (Roche, Basel, Switzerland) and the specific primers are listed in Supplementary Table 1. Then, localization of the total AjCTP1/2 transcripts was detected in the circumoral nervous system. The detailed method used was described by Li et al. (2022). Briefly, the tissue was prehybridized after rehydration and proteinase K treatment, following the addition of antisense RNA probes that bind specifically to mRNA (sense RNA probes were used for negative control). Blocking buffer was used for blocking non-specific binding sites and then alkaline phosphatase-conjugated anti-digoxigenin antibodies were added according to the manufacturer’s instruction, which could specifically bind to the hybridized nucleic acid probe. Finally, the chromogenic substrate NBT/BCIP was added for visualization of AjCTP1/2 expression. Images were taken using an Olympus bright field light microscope (Olympus, Tokyo, Japan).

4.7 Analysis of the in vitro activity of AjCT1 and AjCT2 on preparations from A. japonicus

Longitudinal muscles and intestines (20 mm in length) from A. japonicus were dissected and then suspended in a 50 mL glass organ bath containing 20 mL sterilized seawater. Both ends of the preprations were tied with cotton ligatures and then one end was tied to a metal hook and the other end was connected with a High Grade Isotonic Transducer (ADinstruments MLT0015, Oxford, UK). Before testing, the preparation was stabilized for about 20 min, and the resting tension was adjusted to 1.0 g. Informed by previous findings from Cai et al. (2018), we investigated if AjCT1 and AjCT2 caused relaxation of preparations. First, acetylcholine (ACh) was applied to induce the contraction of preparations and then when a stable state of contraction was achieved, 20 uL AjCT1 or AjCT2 at a range of concentrations was added and relaxation responses were recorded. Output signal (cm) from PowerLab data acquisition hardware (ADinstruments PowerLab 4/26, Oxford, UK) was calculated using LabChart (v8.0.7) software.

4.8 Investigation of the in vivo pharmacological effects of AjCT1 and AjCT2 in A. japonicus

A total of 150 adult sea cucumbers were randomly assigned to five groups (30 individuals per group), including two high concentration groups (CT1H and CT2H, 5 × 10^-1 mg/ml), two low concentration groups (CT1L and CT2L, 5 × 10^-3 mg/ml) and the control group (CO, sterilized seawater), with each group consisting of three parallel experiments comprising ten animals. Before the experiments, sea cucumbers were starved for a day to normalize their physiological status and their wet weights were measured. AjCT1 and AjCT2 were dissolved in sterilized seawater and then injected through the peristome at noon every other day (1 μl/g wet weight) and the CO group were injected in the same way but with sterilized seawater (1 μl/g wet weight). Faeces and residual bait were collected and weighed after drying every day. After 24 days, wet weights of sea cucumbers were measured again. Subsequently, animals were dissected and intestines were collected for detecting the expression levels of the three receptor genes (AjCTR, AjPDFR1, AjPDFR2) and several growth factors, including multiple epidermal growth factor 6 (AjMegf6, GenBank accession number: MG018199.1), insulin-like growth factor (AjIgf, GenBank accession number: PIK50518.1) and growth and differentiation factor-8 (AjGDF-8, GenBank accession number: KP100064.1) (Li et al., 2016; Wang et al., 2018). IBM SPSS Statistics 25.0 was applied for statistical analysis and a p-value < 0.05 was considered as significant difference. The specific primers for qRT-PCR analysis are listed in Supplementary Table 1.

4.9 Loss-of-function experiments by RNA interference (RNAi)

To investigate how receptor expression is affected by knock-down of ArCTP expression, an RNAi experiment was carried out. It was difficult to design AjCTP1- and AjCTP2- specific interference oligonucleotides and therefore, we designed and customized generic interference oligonucleotides targeting both AjCTP1 and AjCTP2 (siAjCTP1/2-1 and siAjCTP1/2-2, containing the extra 2′ ome modification) (Beijing Tsingke Biotech Co., Ltd. China). Meanwhile, a negative control siRNA (siNC) was also customized for the experiment (Supplementary Table 1). The siAjCTP1/2-1, siAjCTP1/2-2 or siNC dry powder was diluted to 20 μM with DEPC water following the manufacturer’s instruction, then 60 μl sterilized seawater was mixed with 20 μl siAjCTP1/2-1, siAjCTP1/2-2 or siNC, and 20 μl Lipo6000 transfection reagent (Beyotime, Shanghai, China) as the transfection solution. Eighteen sea cucumbers were randomly divided into three groups and injected with three different transfection solutions respectively. The mixed solutions were injected through the peristome once a day (1 μl/g wet weight) and intestines were dissected and frozen in liquid nitrogen 3 days after injection. The expression level of AjCTP1/2 was investigated by qRT-PCR and the interference rate was calculated to determine the inhibition efficiency of siRNAs. Finally, the relative expression levels of AjCTR, AjPDFR1 and AjPDFR2 were investigated using the selected A. japonicus intestines.

Competing Interests

The authors declare that no competing interests exist.

Funding

This research was supported by National Natural Science Foundation of China [grant number 42276103].

Author Contributions

Xiao Cong, Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing—original draft preparation; Huachen Liu, Methodology, Validation; Lihua Liu, Methodology, Validation; Maurice R. Elphick, Formal analysis, Validation, Writing—review & editing; Muyan Chen, Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing—review & editing

Supplementary figures

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