CT-type neuropeptide precursors and the candidate receptors in A. japonicus. (A) Alternative splicing schematic of AjCTP1 and AjCTP2. Exons are represented by green rectangles, introns are represented by lines, AjCT1 and AjCT2 are represented by red and orange rectangles respectively, upstream and downstream non-coding regions are represented by gray rectangles. The numbers represent the length of exons or introns. (B) Phylogenetic analysis of AjCTP1 and AjCTP2 and CT-related neuropeptide precursors from other bilaterians. CT-type precursors from deuterostomes are shown in green (echinoderms) and purple (chordates), CT-type precursors from protostomes are shown in pink and VP/OT-type neuropeptide precursors (outgroup) are shown in orange. (C) Phylogenetic analysis of relationships of AjCTR, AjPDFR1 and AjPDFR2 (red letters) with CT-type and PDF-type receptors in other taxa (black letters), using VP/OT-type receptors as an outgroup. CTR/CLR-type receptors are shown in purple, deuterostome PDFRs are shown in yellow, protostome PDFRs are shown in green and VP/OTRs are shown in pink. Full species names and accession numbers are listed in Supplementary Table 3.

Pharmacological characterisation of AjCT1 and AjCT2 as ligands for the A. japonicus receptors AjCTR, AjPDFR1 and AjPDFR2. (A) CRE-driven luciferase activity measured in HEK293T cells transfected with one of the three candidate receptors after exposure to AjCT1or AjCT2 or neuropeptide + DMSO or neuropeptide + H89 (10 μM). The neuropeptide concentration used here was 10-6 M. (B) Measurement of cAMP accumulation after exposure to AjCT1 or AjCT2 (10-9 – 10-5 M). No cAMP elevation is observed in control experiments where HEK293T cells were transfected with empty pcDNA 3.1(+) (black circle). The EC50 values are shown in the figure. Error bars represent SEM for three independent experiments. * indicates a statistically significant difference with p < 0.05.

SRE-driven luciferase activity and receptor internalization in cells transfected with AjCTR, AjPDFR1 or AjPDFR2 and exposed to AjCT1 or AjCT2. (A) SRE-driven luciferase activity measured after incubation with AjCT1, AjCT2, neuropeptide (10-6 M)/DMSO, neuropeptide (10-6 M)/FR900359 (1uM) and neuropeptide (10-6 M)/Gö 6983 (1 μM). (B) Localization and internalization of AjCTR, AjPDFR1 and AjPDFR2 after 15 min treatment with 10-6 M AjCT1 or AjCT2. Green fluorescence represents the localization of pEGFP-N1/receptors in cells. The cell nucleus probe (DAPI) was used for cell nucleus staining. Scale bar: 50 µm. Error bars represent SEM for three independent experiments. * indicates a statistically significant difference with p < 0.05. ns, indicates no significant difference. All pictures are representative of at least three independent experiments.

Activation of signaling pathways

ERK1/2 activity in cells transfected with AjCTR, AjPDFR1 or AjPDFR2 and exposed to AjCT1 or AjCT2. (A) and (B) show the immunoblot intensity of representative phosphorylation bands. The transfected HEK293T cells were incubated with AjCT1 (10-6 M) or AjCT2 (10-6 M) for 0, 5, 15, 30, 45 and 60 min. The p-ERK1/2 was normalized based on t-ERK1/2. Error bars represent SEM for three independent experiments. Statistically significant differences are indicated as follows: *: p < 0.05, **: p < 0.01.

The expression profile of AjCTP1/2 in A. japonicus. (A) Analysis of the transcript expression level of AjCTP1/2 in different tissues. Lowercase letters a and b above columns indicate the statistical difference at p < 0.05; the expression level of AjCTP1/2 in CNS was significantly higher than that in other tissues. Mean values with standard deviations (n = 5) are shown. (B) Localization of AjCTP1/2 in CNS using mRNA in situ hybridization. CNS, circumoral nervous system; CT, connective tissue; Es, epidermis. Scale bar: 100 µm.

Pharmacological effects of AjCT1 and AjCT2 on longitudinal muscle and intestine preparations from A. japonicus. (A) and (B) show representative recordings of the relaxing effects of AjCT1 and AjCT2 on longitudinal muscle preparations. The graphs show the dose-dependent relaxing effects of AjCT1 and AjCT2 (10-10 – 10-6 M), calculated as the percentage reversal of the contracting effect of 10-6 M ACh. (C) and (D) show representative recordings of experiments in which AjCT1 (10-7 – 10-5 M) and AjCT2 (10-9 – 10-5 M) were tested on intestine preparations. AjCT1 had no effect, but AjCT2 caused relaxation and the graph shows the dose-dependent relaxing effect of AjCT2 (10-9 – 10-5 M), calculated as the percentage reversal of the contracting effect of 10-5 M ACh. Mean values ± SEM were determined from three preparations. Lowercase letters a, b and c above columns indicate the statistical difference at p < 0.05, whilst there is no significant difference between bc and c.

In vivo pharmacological tests revealed that AjCT1 and AjCT2 affect feeding and growth-related gene expression in A. japonicus. (A) Body mass in different groups before and after injection of AjCT1, AjCT2 or sterilized seawater. (B) The relative intestinal transcript expression levels of AjGDF-8, AjIgf and AjMegf6 in different groups after injection of AjCT1 or AjCT2 or sterilized seawater (CO) in A. japonicus. (C) The relative intestinal expression levels of AjCT1/2 receptors (AjCTR, AjPDFR1 and AjPDFR2) in CT2H and CO groups after 24 days injection experiment. Values were mean ± SEM (n = 5). * indicates statistically significant differences with p < 0.05, ** indicates statistically significant differences with p < 0.01. CO: control group (sterilized seawater); CT1L/CT2L: AjCT1/AjCT2 low concentration group (5 × 10-3 mg/mL); CT1H/CT2H: AjCT1/AjCT2 high concentration group (5 × 10-1 mg/mL).

The relative intestinal expression levels of AjCT1/2 receptors after AjCTP1/2-1 knockdown. (A), (B) and (C) show the relative expression levels of AjCTR, AjPDFR1 and AjPDFR2 respectively. Values are means ± SEM (n = 5). ** indicates statistically significant differences with p < 0.01.

Summary of CT-related neuropeptides and their corresponding precursors, receptors, RAMPs and feeding/growth regulation in representative species. Echinoderms are boxed in purple dotted lines. The summary of A. japonicus is framed in red dotted lines. A green rectangle indicates the presence of neuropeptides or precursors. The red pentagram indicates that neuropeptides are generated by alternative splicing of precursor transcripts, unfilled pentagram represents a single gene encoding one neuropeptide, the filled pentagram represents a gene encoding two neuropeptides in tandem. For the receptors for CT, pink filled rectangles show that the presence of the corresponding receptors has been reported and unfilled rectangles indicate that their receptors have not been reported. Blue filled rectangles indicate that RAMPs have been identified in this species, blue rectangles with a line show that RAMPs have not been identified in this species. Yellow filled rectangles indicate that feeding/growth regulation by CT-type neuropeptides has been investigated and unfilled rectangles showed that there was no relevant study in this species. I: Inhibition; P: Promotion; N: Ineffective.

Calcitonin-type (CT-type) neuropeptide precursors in A. japonicus. (A) AjCTP1 comprises two putative CT-type neuropeptides, AjCT1 and AjCT2, whereas AjCTP1 generated by alternative splicing comprises only AjCT2. The predicted signal peptide is shown in blue, the predicted cleavage sites (KR or KK) are shown in green, CT-type neuropeptides are shown in red and C-terminal glycines that are potential substrates for amidation are shown in orange. Predicted disulfide bridges in AjCT1 and AjCT2 are formed between the two underlined cysteines. (B) Nucleotide sequences encoding CT-type neuropeptide precursors and the deduced amino acid sequences in A. japonicus. 3’UTR and 5’UTR were shown in lowercase and ORF was shown in uppercase. Blue uppercase labeled the predicted signal peptide, the green uppercase showed the predicted cleavage site, AjCT1 and AjCT2 were shown in red and the putative substrate for amidation was shown in orange. (C) The tertiary structures of AjCTP1 and AjCTP2 as predicted by SWISS-MODEL

Comparison of CT-type neuropeptides from Bilateria: deuterostomes-ambulacraria (green), chordata (purple) and protostomes (pink). The conserved residues are shown with white lettering highlighted in gray and black. The accession numbers or citations are shown in the Supplementary Table 2.

Amino acid sequences and phosphorylation sites of AjCTR, AjPDFR1 and AjPDFR2 in A. japonicus. (A), (B) and (C) show the cDNA sequences encoding the receptors and their deduced amino acid sequences. ORFs are shown in uppercase; 3’UTR and 5’UTR are shown in lowercase. The stop codon is shown by the asterisk (*). Predicted potential phosphorylation sites are shown with squares.

Characterization of AjCT1 and AjCT2 receptors (AjCTR, AjPDFR1 and AjPDFR2). (A) Conserved domains of CTR/CLR-type protein sequences. Black line represents the length of proteins and different domains are represented by rectangles. (B) Conserved domains of PDFRs in Bilateria. Black line represents the length of proteins and different domains are represented by rectangles. (C) Synteny analysis of AjCT1 and AjCT2 receptors (AjCTR, AjPDFR1 and AjPDFR2). The chromosomal locations of AjCT1 and AjCT2 receptor genes from A. rubens, L. variegatus and A. japonicus are shown, with different colors used to mark neighboring genes and with their transcriptional direction shown by arrows. GOLGA4, golgin subfamily A member 4; NeuroD, neurogenic differentiation factor; CRF2, corticotropin-releasing factor receptor 2; CTR, calcitonin type receptor; CLR, calcitonin gene-related peptide type 1 receptor; ppGalNAc-T, polypeptide N-acetylgalactosaminyltransferase; URB3, E3 ubiquitin-protein ligase ubr3; SLC40A1, solute carrier family 40 member 1; CMAH, cytidine monophosphate-N-acetylneuraminic acid hydroxylase; MAGP, microfibril-associated glycoprotein; HCN, hyperpolarization-gated and cyclic nucleotide regulated channel; ATG9A, autophagy-related protein 9A-like; lysRS, lysine--tRNA ligase; FCN, ficolin; PDFR1, pigment-dispersing factor receptor 1; PDFR2, pigment- dispersing factor receptor 2; PSMD8, 26S proteasome regulatory subunit 8; SARNP, SAP domain-containing ribonucleoprotein; HPM1, histidine protein methyltransferase 1; PNPO, pyridoxine-5’-phosphate oxidase.

Localization of AjCTR, AjPDFR1 and AjPDFR2 . Green fluorescence represents the localization of pEGFP-N1/receptors in cell membrane. Cell nucleus probe (DAPI) was applied for cell nucleus staining.

Effects of PKA or PKC inhibitor on AjCT1 or AjCT2 stimulated ERK1/2 phosphorylation in AjCTR, AjPDFR1 or AjPDFR2 expressing HEK293T cells. (A) ERK1/2 phosphorylation, activated by AjCT1, is blocked by PKC inhibitor Gö 6983 in AjCTR expressing HEK293T cells and is blocked by PKA inhibitor H89 and PKC inhibitor in AjPDFR1 expressing HEK293T cells. (B) ERK1/2 phosphorylation, activated by AjCT2, is blocked by PKC inhibitor in AjCTR expressing HEK293T cells and is blocked by PKA inhibitor and PKC inhibitor in AjPDFR1 and AjPDFR2 expressing HEK293T cells. Serum-starved HEK293 cells were pre-treated with DMSO, PKA inhibitor, or PKC inhibitor before AjCT1 or AjCT2 stimulation. All pictures and data are representative for at least three independent experiments.

Negative control of circumoral nervous system in situ hybridization. The sense probe for AjCTP1/2 was incubated with sections of tissue from adult A. japonicus instead of an antisense probe. Scale bar: 100 µm.

The mass of remaining bait and the excrement. (A) Calculation of the remaining bait mass in different phases. (B) The mass of excrement in different phases. Every four days was divided into one phase (I-VI) for statistics. CO: control group (sterilized seawater); CT2L: AjCT2 low concentration group (5 × 10-3 mg/mL); CT2H: AjCT2 high concentration group (5 × 10-1 mg/mL).

The relative expression level of AjCTP1/2 in siNC, siAjCTP1/2-1 and siAjCTP1/2-2. Values are means ± SEM (n = 5). Asterisks represent the significant difference as follows: *: p < 0.05, **: p < 0.01. siNC: negative control group; siAjCTP1/2-1 and siAjCTP1/2-2: experimental groups targeting AjCTP1/2.