Introduction {#s1}
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Nervous systems, from simple nerve nets in primitive species to complex architectures in vertebrates, process sensory stimuli and enable animals to generate body-wide responses ([@bib2]). Neurosecretory centers, one of the major output systems in the animal brain, secrete neuropeptides and nonpeptidergic neuromodulators to regulate developmental, behavioral and physiological processes ([@bib57]). Understanding the evolutionary origin of these centers is an area of active investigation, mostly because of their importance in a range of physical phenomena such as growth, metabolism, or reproduction ([@bib58]; [@bib66]).

The hypothalamus constitutes the major part of the ventral diencephalon in vertebrates and acts as a neurosecretory brain center, controlling the secretion of various neuropeptides (hypothalamic neuropeptides) ([@bib3]; [@bib5]). Beyond vertebrates, similar neurosecretory systems have been seen in multiple protostomian species, including crustaceans, spiders, and mollusks ([@bib18]). Specific to echinoderms, which occupy an intermediate phylogenetic position asdeuterostomian invertebrate species with respect to vertebrates and protostomes, increasing evidence, collected from in silico identification of hypothalamic neuropeptides and the functional characterization of a vasopressin/oxytocin (VP/OT)-type signaling system, suggests the existence of conserved signaling elements ([@bib66]; [@bib37]).

The hypothalamic neuropeptide kisspeptins, encoded by the *Kiss1* gene and most notably expressed in the hypothalamus, share a common Arg-Phe-amide motif at their C-termini and belong to the RFamide peptide family ([@bib44]; [@bib59]). Exogenous administration of kisspeptins triggers an increase in the circulating levels of gonadotropin-releasing hormone and gonadotropin in humans, mice, and dogs ([@bib16]; [@bib11]; [@bib12]; [@bib1]). Accumulating evidence suggests that the kisspeptin system functions as a central modulator of the hypothalamic-pituitary-gonadal (HPG) axis to regulate mammalian puberty and reproduction through a specific receptor, GPR54 (also known as AXOR12 or hOT7T175), which is currently referred to as the kisspeptin receptor ([@bib34]; [@bib25]; [@bib21]). Following the discovery of kisspeptins and kisspeptin receptors in mammals, a number of kisspeptin-system paralogous genes have been revealed in other vertebrates ([@bib40]), and a couple of functional kisspeptin and its receptor have also been demonstrated in amphioxus ([@bib63]). Moreover, kisspeptin-type peptides and their corresponding receptors from echinoderms have been annotated in silico, on the basis of the analysis of genome and transcriptome sequence data ([@bib32]; [@bib14]; [@bib45]; [@bib46]; [@bib55]; [@bib8]). However, to our knowledge, neither the kisspeptin-type peptides nor the corresponding receptors have been experimentally identified and functionally characterized in non-chordate invertebrates. This raises an important question: does the kisspeptin signaling system have an ancient evolutionary origin or did it evolve de novo in the chordate/vertebrate lineages?

Here, we addressed this question by searching for kisspeptin and its receptor genes in a non-chordate species, the sea cucumber *Apostichopus japonicus*. It is one of the most studied echinoderms and is widely distributed in temperate habitats in the western North Pacific Ocean, being cultivated commercially on a large scale in China ([@bib43]). We uncovered kisspeptin-like and kisspeptin receptor-like genes by mining published *A. japonicus* data ([@bib67]; [@bib8]) using a bioinformatics approach. Their signaling properties were characterized using an in vitro culture system. Through the evaluation of Ca^2+^ mobilization and other intracellular signals, we found that *A. japonicus* kisspeptins activate two receptors (AjKissR1 and AjKissR2) via a GPCR-mediated G~αq~/PLC/PKC/MAPK signaling pathway in a mammalian cell line. Although likely, it remains to be determined whether the same signaling cascade also occurs in vivo in its seemingly conserved function in reproductive control. Finally, we revealed the physiological activities of this signaling system both in vivo and ex vivo, and we demonstrated the involvement of the kisspeptin system in reproductive and metabolic regulation in *A. japonicus*. Collectively, our findings indicate the existence of a kisspeptin signaling system in non-chordate deuterostome invertebrates and provide new evidence to support the ancient evolutionary origin of the intracellular signaling and physiological functions mediated by this kisspeptin system ([@bib58]).

Results {#s2}
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In silico identification of kisspeptins and kisspeptin receptors {#s2-1}
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The putative *A. japonicus* kisspeptin precursor gene (*AjpreKiss*) was identified in silico from transcriptome data and cloned from anterior part (ANP, containing the nerve ring) tissue samples by reverse transcription polymerase chain reaction (RT-PCR). The full-length cDNA (GenBank accession number MH635262) was 2481-bp long and contained a 543-bp open reading frame (ORF), encoding a 180-amino-acid peptide precursor (AjpreKiss) with one predicted signal peptide region and four cleavage sites ([Figure 1A](#fig1){ref-type="fig"} and [Figure 1---figure supplement 1](#fig1s1){ref-type="fig"}). Two mature peptides with amide donors for C-terminal amidation---a 32-amino-acid kisspeptin-like peptide with a disulﬁde-bond (AjKiss1a) and an 18-amino-acid kisspeptin-like peptide (AjKiss1b)---were predicted (sequences listed in the \'Key Resources Table\'). The organization of the *AjpreKiss* gene as determined from the genome, as well as those of the zebrafish *DrpreKiss1*, *DrpreKiss2* and human *HspreKiss1*, showed two exons in the ORF region ([Figure 1B](#fig1){ref-type="fig"}). Alignment of multiple sequences revealed a high similarity between AjKiss1a/b and predicted echinoderm kisspeptins, but low identity between AjKiss1a/b and vertebrate kisspeptin 1/2 ([Figure 1C](#fig1){ref-type="fig"}). A maximum likelihood tree of kisspeptin precursors, including PrRP, 26RFa/QRFP, GnIH, and NPFF from outgroups ([@bib60]), was constructed for phylogenetic analysis. It showed that *AjpreKiss*, together with kisspeptin-like precursor genes from the sea cucumbers *Holothuria scabra* and *H. glaberrima*, were grouped with the vertebrate kisspeptin 1 and kisspeptin 2 subfamilies into the 'Kisspeptin' group ([Figure 1D](#fig1){ref-type="fig"}).

![Gene structure, homology, and phylogenetic characterization of *Apostichopus japonicus* kisspeptin precursor (AjpreKiss).\
(**A**) Deduced amino-acid sequence of AjpreKiss. The signal peptide is labeled in the box with full lines; the cleavage sites are highlighted in red; glycine residues responsible for C-terminal amidation are highlighted in green; cysteines paired in a disulfide-bonding structure are highlighted in light blue; the predicted mature peptides, AjKiss1a and AjKiss1b, are noted by the blue and green underlines. (**B**) The organization of the *AjpreKiss* gene is compared with the zebrafish and human *preKiss* genes. The exon-intron data were obtained from the respective genomic sequences from NCBI (MRZV01001091.1, NC_007122.7, NC_007115.7 and NC_000001.11). DNA structure is shown with exons numbered in green bands. ATG represents the start methionine codon and TGA represents the stop codon. (**C**) Alignment of the predicted echinoderm kisspeptin core sequences and functionally characterized chordate kisspeptins. Sequences of *Holothuria scabra*, *Holothuria glaberrima, Strongylocentrotus purpuratus,* and *Asterias rubens* kisspeptins were predicted by Elphick's lab ([@bib46]; [@bib55]). Vertebrate kisspeptin core sequences were obtained from GenBank with detailed sequences listed in [Figure 1---source data 1](#fig1sdata1){ref-type="supplementary-material"}. The color align property was generated using Sequence Manipulation Suite online. The percentage of sequences that must agree for identity or similarity coloring was set as 40%. (**D**) Phylogenetic tree of the kisspeptin precursor and four different neuropeptide outgroups ([@bib32]). The tree was constructed on the basis of approximately Maximum-Likelihood algorithms using FastTree two with pre-trimmed sequences. Local support values (%) were provided using the Shimodaira-Hasegawa (SH) test and are indicated by numbers at the nodes. The detailed complete sequences are listed in [Figure 1---source data 2](#fig1sdata2){ref-type="supplementary-material"} and trimmed sequences are listed in [Figure 1---source data 3](#fig1sdata3){ref-type="supplementary-material"}.\
Figure 1---source data 1.Core sequences of kisspeptin from multiple species for alignment.\
Figure 1---source data 2.Amino-acid sequences of the kisspeptin precursor and outgroups for phylogenetic analysis.\
Figure 1---source data 3.Trimmed sequence alignment for phylogenetic tree construction.](elife-53370-fig1){#fig1}

Several predicted 'G-protein coupled receptor 54-like' or 'kisspeptin receptor-like' gene annotations in the hemichordate *Saccoglossus kowalevskii* (two genes), the echinoderm *Acanthaster planci* (two genes), and *S. purpuratus* (seven genes) have been reported ([@bib13]; [@bib50]; [@bib17]). Using these predicted genes as reference sequences to search the *A. japonicus* genomic database, three *A. japonicus* kisspeptin receptor-like genes (*AjKissR1, AjKissR2*, and *AjKissRL3*; GenBank accession numbers, MH709114, MH709115, and MG199220, respectively) were identified and cloned from an *A. japonicus* ovary by RT-PCR. The ORFs of both *AjKissR1* and *AjKissR2* comprised three exons ([Figure 2A](#fig2){ref-type="fig"}), with deduced amino acid sequences of 378 and 327 residues, and contained seven transmembrane domains ([Figure 2B](#fig2){ref-type="fig"} and [Figure 2---figure supplement 1](#fig2s1){ref-type="fig"}). (Detailed data for another putative receptor *AjKissRL3* have not been presented because it exhibited no interaction with ligands in further experiments.) Sequence alignment of AjKissR1 and AjKissR2 with the well-characterized chordate GPR54 was performed ([Figure 2---figure supplement 2](#fig2s2){ref-type="fig"}) and a relatively high identity in seven-transmembrane region sequences, against 21 vertebrate GPR54 sequences, was observed (as shown in [Figure 2---figure supplement 3](#fig2s3){ref-type="fig"}). Maximum likelihood phylogenetic tree analysis, using 'Allatostatin-A receptor' and 'Galanin receptor' as outgroups, revealed that AjKissR1 and AjKissR2 both clustered in the 'Kisspeptin receptor' group. AjKissR1 grouped with *S. purpuratus* (sea urchin) kisspeptin receptors (XP_784787.2, XP_796690.2, XP_793873.2 and XP_796286.1) and hemichordate *S. kowalevskii* (acorn worm) kisspeptin receptor (NP001161574.1), whereas AjKissR2 clustered with the predicted *A. planci* (starfish) Kisspeptin receptors (Genbank ID: XP_022096858.1 and XP_022096775.1) and with the *S. purpuratus* kisspeptin receptor (XP_003727259.1) ([Figure 2C](#fig2){ref-type="fig"}).

![Gene structure and phylogenetic characterization of *Apostichopus japonicus* kisspeptin receptors (AjKissR1 and AjKissR2).\
(**A**) DNA structures of AjKissR1 and AjKissR2. *AjKissR1/2* DNA structure is shown with exons numbered in green bands. ATG represents the start methionine codon and TGA/TAG represents the stop codon. (**B**) Organization of the predicted protein structures. The seven transmembrane domains (TM1--TM7) are marked with red boxes. The N-terminal region and three extracellular (EC) rings are noted with blue boxes, and the C-terminal part and three intracellular (IC) rings are indicated with black boxes. Stop codons are represented by an asterisk. Arabic numbers under the bands indicate the nucleotide or amino acid sites. (**C**) Phylogenetic tree for kisspeptin, allatostatin-A and galanin receptors (KissR, AllaR and GalaR). The tree was constructed on the basis of approximately Maximum-Likelihood algorithms by FastTree two using AllaRs and GalaRs as outgroups ([@bib60]). Local support values were provided using the Shimodaira-Hasegawa (SH) test. The detailed sequences are listed in [Figure 2---source data 1](#fig2sdata1){ref-type="supplementary-material"}.\
Figure 2---source data 1.Amino-acid sequences of kisspeptin receptors and outgroups for phylogenetic analysis.](elife-53370-fig2){#fig2}

Functional expression of putative kisspeptin receptors {#s2-2}
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To verify the exact expression and localization of the putative *A. japonicus* kisspeptin receptors, AjKissR1 and AjKissR2 with an N-terminal FLAG-tag or with enhanced green fluorescent protein (EGFP) fused to the C-terminal end were constructed and stably or transiently expressed in human embryonic kidney 293 (HEK293) cells, respectively. As shown in [Figure 3A](#fig3){ref-type="fig"}, AjKissR1 and AjKissR2 were predominantly expressed and localized on the surface of HEK293 cells, with some intracellular accumulation, in the absence of the ligand. Next, to examine whether AjKissR1 and AjKissR2 are activated by synthetic kisspeptins, a Ca^2+^ mobilization assay based on the calcium probe Fura 2 was performed. As shown in [Figure 3B](#fig3){ref-type="fig"}, both AjKiss1a and AjKiss1b elicited a rapid increase of intracellular Ca^2+^, in a concentration-dependent manner, in HEK293 cells transfected with AjKissR1 and AjKissR2. However, AjKissR1 was preferentially activated by AjKiss1b, with an EC50 value of 8.06 nM ([Figure 3B 2](#fig3){ref-type="fig"}), whereas AjKissR2 was more specifically activated by AjKiss1a, with an EC50 value of 1.98 nM ([Figure 3B 1](#fig3){ref-type="fig"}).

![Functional characteristics of *Apostichopus japonicus* kisspeptins and receptors.\
(**A**) The cells transiently expressing AjKissR1-EGFP or AjKissR2-EGFP were stained with cell membrane probe (DiI) and cell nucleus probe (DAPI) and detected by confocal microscopy. (**B**) After loading with Fura-2/AM, HEK293 cells expressing either FLAG-AjKissR1 or FLAG-AjKissR2 were exposed to the indicated concentrations of AjKiss1a (B1) and AjKiss1b (B2), and continuous fluorescence was recorded. AUC, Area Under the Curve. [Figure 3B---source data 1](#fig3sdata1){ref-type="supplementary-material"} shows the primary metadata. (**C**) Internalization of AjKissR1-EGFP or AjKissR2-EGFP initiated by 1.0 μM of the indicated ligand was determined after a 60 min incubation by confocal microscopy. All pictures and data are representative of at least three independent experiments.\
Figure 3---source data 1.Primary metadata of Ca^2+^ mobilization assay for [Figure 3B](#fig3){ref-type="fig"}.](elife-53370-fig3){#fig3}

Agonist-mediated receptor internalization from the cell surface into the cytoplasm has been recognized as a key mechanism in regulating the strength and duration of GPCR-mediated cell signaling and directly reflects the activation of the receptor ([@bib49]; [@bib33]). In this study, C-terminal fusion expression of AjKissR1 and AjKissR2 with EGFP was used to track the internalization and trafficking of receptors. As shown in [Figure 3C](#fig3){ref-type="fig"}, AjKissR1 and AjKissR2 were activated by AjKiss1b and AjKiss1a, respectively, to undergo significant internalization from the plasma membrane to the cytoplasm. These data provide clear evidence that AjKissR1 and AjKissR2 are functional receptors that are specific for neuropeptides AjKiss1b and AjKiss1a, respectively.

Ligand selectivity of *A. japonicus* kisspeptin receptors {#s2-3}
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To examine the cross-activity of *A. japonicus* and vertebrate kisspeptin receptors, we detected the potential of synthetic *A. japonicus*, human, frog, and zebrafish kisspeptins (HsKiss1-10, XtKiss1b-10, DrKiss1-10, and DrKiss2-10) in triggering intracellular Ca^2+^ mobilization. As indicated in [Figure 4](#fig4){ref-type="fig"}, HsKiss1-10 and XtKiss1b-10 exhibited higher potency for AjKissR1, whereas both DrKiss1-10 and DrKiss2-10 showed much lower potency in eliciting Ca^2+^ mobilization ([Figure 4A](#fig4){ref-type="fig"}). For the activation of AjKissR2, however, XtKiss1b-10, DrKiss1-10, and DrKiss2-10 had a higher potency than that of HsKiss1-10 ([Figure 4B](#fig4){ref-type="fig"}). However, human neuropeptide S (HsNPS) showed no potency in activating either AjKissR1 or AjKissR2 ([Figure 4C and D](#fig4){ref-type="fig"}). Further analysis demonstrated that both AjKiss1a and AjKiss1b could activate HsKiss1R, DrKiss1Ra, and DrKiss1Rb with different potency ([Figure 4E, F and G](#fig4){ref-type="fig"}).

![Functional cross-activity between the *A. japonicus* and vertebrate Kisspeptin/Kisspeptin receptor systems.\
Intracellular Ca^2+^ mobilization in AjKissR1- ([**A**]{.smallcaps}) or AjKissR2-expressing (**B**) HEK293 cells was measured in response to 1.0 μM DrKiss1-10, DrKiss2-10, XtKiss1b-10, or HsKiss1-10 using Fura-2/AM. No Ca^2+-^mobilization-mediated activity was detected in AjKissR1- (**C**) or AjKissR2-expressing (**D**) HEK293 cells upon administration of the indicated concentrations of human neuropeptide S (HsNPS). Intracellular Ca^2+^ mobilization in human kisspeptin receptor (HsKiss1R)-expressing HEK293 cells was measured in response to 1.0 μM HsKiss1-10, AjKiss1a or AjKiss1b (**E**), as well as in zebrafish kisspeptin receptor (DrKiss1Ra or DrKiss1Rb)-expressing cells responding to 1.0 μM DrKiss1-10, AjKiss1a, or AjKiss1b (**F, G**). [Figure 4---source data 1](#fig4sdata1){ref-type="supplementary-material"} shows the primary metadata. All data shown are representative of at least three independent experiments.\
Figure 4---source data 1.Primary metadata of Ca^2+^ mobilization assay for [Figure 4A-G](#fig4){ref-type="fig"}.](elife-53370-fig4){#fig4}

*A. japonicus* kisspeptin receptors are directly activated by kisspeptins via a G~αq~-dependent pathway {#s2-4}
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Previous studies have demonstrated that in mammals, Kiss1R couples to G~αq~ protein, triggering phospholipase C (PLC), intracellular Ca^2+^ mobilization, and the PKC signaling cascade in response to agonists ([@bib7]). To elucidate G protein coupling in the activation of both AjKiss1a and AjKiss1b, a combination of functional assays, with different inhibitors, was performed. As shown in [Figure 5A](#fig5){ref-type="fig"}, AjKiss1a- and AjKiss1b-eliciting Ca^2+^ mobilization through receptors AjKissR1 and AjKissR2, respectively, was completely blocked by pre-treatment with FR900359, a specific inhibitor of G~αq~ protein ([@bib27]), and also significantly attenuated by PLC inhibitor U73122, the extracellular calcium chelator EGTA, and intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane N,N,N\',N\'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) ([@bib48]). By contrast, the G~αi~ protein inhibitor Pertussis Toxin (PTX) showed no effect on the Ca^2+^ mobilization activated by *A. japonicus* kisspeptins in AjKissR1- or AjKissR2-expressing cells. These data indicate the involvement of G~αq~ protein in the AjKissR1- and AjKissR2-mediated intracellular signaling pathway.

![*Apostichopus japonicus* kisspeptin receptors are directly activated by kisspeptins via a G~αq~-dependent pathway.\
(**A**) Intracellular Ca^2+^ mobilization in AjKissR1- and AjKissR2-expressing HEK293 cells was measured in response to 100 nM AjKiss1a or AjKiss1b, using cells that had been pre-treated for 12 hr with G~αi~ protein inhibitor (PTX, 100 ng/mL), or for 1 hr with DMSO, G~αq~ protein inhibitor (FR900359, 1.0 μM), PLC inhibitor (U73122, 10 μM), intracellular calcium chelator (BAPTA-AM, 100.0 μM), or extracellular calcium chelator (EGTA, 5.0 mM). [Figure 5---source data 1](#fig5sdata1){ref-type="supplementary-material"} presents the primary metadata. Pictures shown are representative of at least three independent experiments. (**B**) Competitive binding of 1.0 μM fluorescein isothiocyanate (FITC)-AjKiss1a to AjKissR1 or AjKissR2 in the presence of the indicated concentration of AjKiss1a or AjKiss1b. Error bars represent the SEM for at least three independent experiments.\
Figure 5---source data 1.Primary metadata of Ca^2+^ mobilization assay and binding assay for [Figure 5A and B](#fig5){ref-type="fig"}.](elife-53370-fig5){#fig5}

Next, a competitive binding assay was established by using a synthesized AjKiss1a that had a fluorescein isothiocyanate (FITC) tag at the N-terminus (FITC--AjKiss1a, sequence listed in the \'Key Resources Table\') to assess the direct interaction of AjKissR1 and AjKissR2 with AjKiss1a and AjKiss1b. Functional assays revealed that FITC--AjKiss1a exhibited the potential to induce Ca^2+^ mobilization that was comparable to that induced by the wild-type neuropeptide ([Figure 5---figure supplement 1](#fig5s1){ref-type="fig"}). The competitive displacement of FITC--AjKiss1a with AjKiss1a and AjKiss1b in HEK293/AjKissR1 and HEK293/AjKissR2 cells was measured by FACS (fluorescence-activated cell sorting) analysis. As shown in [Figure 5B](#fig5){ref-type="fig"}, unlabeled AjKiss1a and AjKiss1b were found to compete with FITC-labeled AjKiss1a with IC~50~ values of 95.16 and 353.30 nM in AjKissR2- and AjKissR1-transfected HEK293 cells, respectively.

AjKissR1 and AjKissR2 are activated by AjKiss1b-10 and signal through the G~αq~-dependent MAPK pathway {#s2-5}
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As AjKiss1b-10 exhibited high potency in activating both AjKissR1 and AjKissR2 in HEK293 cells ([Figure 6---figure supplement 1](#fig6s1){ref-type="fig"}), it was used to conduct further in vitro and in vivo experiments. The previous results reveal that AjKissR1 and AjKissR2 can be activated by ligands and signals through G~αq~-dependent Ca^2+^ mobilization; however, the detailed signaling pathway remained to be elucidated. To address this, different inhibitors were used to test intracellular ERK1/2 activation in HEK293 cells expressing AjKissR1 and AjKissR2, treated with Ajkiss1b-10. As shown in [Figure 6A](#fig6){ref-type="fig"}, stimulation with AjKiss1b-10 led to the activation of both AjKissR1 and AjKissR2, inducing significant ERK1/2 activation. Further assessment demonstrated that AjKissR1- or AjKissR2-mediated activation of ERK1/2 was significantly blocked by the PLC inhibitor U73122 (10 μM) and by the PKC inhibitor Gö6983 (1.0 μM) ([Figure 6B and C](#fig6){ref-type="fig"}). Moreover, using a PKC subtype translocation assay, we determined that PKCα, PKCβI, and PKCβII are involved in the activation of the MAPK pathway ([Figure 6D and E](#fig6){ref-type="fig"}). Overall, these results suggest that AjKissR1 and AjKissR2, once activated by ligand, can activate the G~αq~ family of heterotrimeric G proteins, leading to dissociation of the G protein subunits G~βγ~ and the activation of PLC, leading to intracellular Ca^2+^ mobilization. This, in turn, activates PKC (isoform α and β) and the MAPK cascade, particularly ERK1/2, via the G~αq~/PLC/PKC signaling pathway ([Figure 6F](#fig6){ref-type="fig"}).

![ERK1/2 activation mediated by AjKissR1 or AjKissR2.\
(**A**) Concentration- and time-dependent effects of AjKiss1b-10 on ERK1/2 phosphorylation in HEK293 cells that were stably expressing FLAG--AjKissR1 or FLAG--AjKissR2. Cells were challenged with different concentrations of AjKiss1b-10 for 5 min or incubated with 1.0 μM AjKiss1b-10 for the indicated times. Immunoblots were quantified using a Bio-Rad Quantity One Imaging system. (**B, C**) ERK1/2 phosphorylation, activated by AjKiss1b-10, was blocked by PLC or PKC inhibitors. Serum-starved HEK293 cells expressing FLAG--AjKissR1 or FLAG--AjKissR2 were pre-treated with DMSO, PLC inhibitor (U73122, 10 μM), or PKC inhibitor (Gö6983, 10 μM) for 1 hr prior to AjKiss1b-10 stimulation (1.0 μM). (**D, E**) Role of various PKC isoforms in the activated signaling pathways of the sea cucumber kisspeptin receptor. HEK293 cells, co-transfected with FLAG--AjKissR1 or FLAG--AjKissR2 and different PKC-EGFP isoforms, were stimulated by 1.0 μM AjKiss1b-10 for the indicated times and then examined by confocal microscopy. Red arrows denote the recruitment of PKC--EGFP isoforms on the cell membrane. (**F**) Schematic diagram of agonist-induced *A. japonicus* kisspeptin receptor activation. AjKiss1b-10 binding to AjKissR1 or AjKissR2 activates the G~αq~ family of heterotrimeric G proteins, which leads to dissociation of the G protein subunits G~βγ~, and activates PLC, leading to intracellular Ca^2+^ mobilization. This, in turn, activates PKC (isoform α and β) and stimulates the phosphorylation of ERK1/2. The ratio of p-ERK1/2 to total ERK1/2 was normalized to the peak value detected in the corresponding experiments (for example, the peak value of the ratio of AjKissR1/AjKiss1b-10 (10 μM) for the dose-dependent analysis, or of AjKissR1/AjKiss1b-10 (1.0 Aj) at 5 min for the time-course analysis). All pictures and data are representative of at least three independent experiments.](elife-53370-fig6){#fig6}

Physiological functions of the kisspeptin signaling system in *A. japonicus* {#s2-6}
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To further assess the physiological roles of the kisspeptin signaling system in *A. japonicus*, we examined the tissue distribution of *A. japonicus* kisspeptin and its receptor, using custom rabbit polyclonal antibodies (anti-AjKiss1b-10 and anti-AjKissR1 \[details shown in the \'Key Resources Table\'\]; antibody specificities were evaluated as shown in [Figure 7---figure supplement 1](#fig7s1){ref-type="fig"}). Tissue-specific western blot analysis revealed the expression of the kisspeptin precursor in the respiratory tree (RET), ovary (OVA), testis (TES), and anterior part (ANP, containing the nerve ring as shown in [Figure 7---figure supplement 2E,F](#fig7s2){ref-type="fig"}) of mature sea cucumbers (the maturity of the gonads was evaluated by H and E staining, as shown in [Figure 7---figure supplement 2B](#fig7s2){ref-type="fig"}). AjKissR1 was detected in the RET, OVA, ANP, and muscle (MUS) ([Figure 7A](#fig7){ref-type="fig"}). However, the failure to detect mature peptide fragment using anti-AjKiss1b-10 indicates that the further development of antibodies with high sensitivity and specificity might be required to clarify location of the mature kisspeptin in *A. japonicus* tissues.

![Physiological function analysis of kisspeptin signaling systems in *Apostichopus japonicus*.\
(**A**) Western Blot analysis of *A. japonicus* kisspeptin precursor and kisspeptin receptor (AjKissR1) in different tissues of sea cucumber. INT, intestine; RET, respiratory tree; ANP, anterior part; OVA, ovary; TES, testis; MUS, muscle; and BOW, body wall. (**B**) Immunofluorescence histochemical staining of *A. japonicus* kisspeptin precursor and AjKissR1 in RET, OVA, TES, MUS (B1) and nerve ring (B2) of the sea cucumber. CE, coelomic epithelium; BB, brown body; CM, cell membrane; SE, spermatogenic epithelium; EM, epithelium of muscle; OS, outer surface; and IR, internal region. (**C**) ERK1/2 phosphorylation activity of kisspeptins and the inhibitory effect of a vertebrate kisspeptin antagonist (pep234) on the cultured ovary of sea cucumber. Samples were collected and fixed after 2 hr of ligand administration with or without a 4-hr pre-treatment with pep234, in optimized L15 medium at 18°C. Error bars represent SEM for three independent experiments. Immunoblots were quantified using a Bio-Rad Quantity One Imaging system. (**D**) Immunofluorescence histochemical staining of p-ERK signal in cultured oocytes of sea cucumber. Samples were collected and fixed after 2 hr of ligand administration with or without a 4-hr pretreatment with pep234, in optimized L15 medium at 18°C. NC indicates the nucleus of oocytes. (**E, F**) Variation of body weight and tissue index (ratio of tissue weight/body weight) over 40 days of stimulus treatment. Each symbol and vertical bar represent mean ± SEM (n = 5 animals). \*\* indicates extremely significant differences (p=0.0001), as demonstrated by one-way ANOVA followed by Tukey's multiple comparisons test. (**G**) Degenerated intestine in AjKiss1b-10 treated sea cucumbers. (**H**) Heatmap showing the expression profile of *A. japonicus* kisspeptin and kisspeptin receptors (*AjKissR1/R2* and *AjKiss1*) in different tissues and developmental stages of sea cucumber. The variation in color represents the relative expression level of each gene in different samples (normalized against the peak values in all samples and logarithmized). The number of animals used for all samples is six, except for the number of ovary samples, with one in NOV (November) and JUN (June), three in DEC (December) and FEB (February), five in MAR (March), and six in APR (April) and MAY (May), and in testis, with two in NOV (November) and DEC (December), four in FEB (February) and MAR (March), and six in APR (April) and MAY (May). [Figure 7---source data 1](#fig7sdata1){ref-type="supplementary-material"} represents the primary metadata. All pictures and data are representative of at least three independent experiments.\
Figure 7---source data 1.Primary metadata of body weight, tissue index and qPCR assay for [Figure 7E, F and H](#fig7){ref-type="fig"}.](elife-53370-fig7){#fig7}

To reveal the in situ distribution of the kisspeptin precursor and receptor, we performed immunofluorescence labeling on tissue sections. Consistent with results from the western blot assay, significant expression of the kisspeptin precursor was observed in the RET, TES, and nerve ring in the ANP sections, with no expression in the MUS and OVA (the inconsistency with results from the western blotting assay vs. immunofluorescence may be due to either differences in the specific structures and components of oocytes or differences in the characteristics of the antibody); AjKissR1 expression was observed in the RET, OVA, MUS and nerve ring in ANP sections, with rare expression in TES ([Figure 7B](#fig7){ref-type="fig"}). At the cellular level, the kisspeptin precursor was mainly detected in the coelomic epithelium of RET, whereas AjKissR1 was detected in the brown bodies, which can be found in the luminal spaces of RET and might be related to foreign material removal ([@bib51]). In particular, significant expression and cell membrane localization of AjKissR1 was detected in oocytes, indicating the consistent molecular property of AjKissR1 in vivo and in vitro. From the TES sections, significant fluorescence signal from the kisspeptin precursor, but only a weak signal from AjKissR1, can be detected in spermatogenic epithelium. A significant expression of AjKissR1 was detected in the epithelium of muscle from MUS sections. Moreover, in the ANP sections, the kisspeptin precursor was detectable in the outer surface part of the nerve ring (mainly containing the cell bodies of neurons, as shown in [Figure 7---figure supplement 2 F2](#fig7s2){ref-type="fig"}), whereas the AjKissR1 was detected in the internal region of the nerve ring (mainly containing the axons of neurons, as shown in [Figure 7---figure supplement 2 F2](#fig7s2){ref-type="fig"}). The distribution profile of *A. japonicus* kisspeptin and kisspeptin receptor indicates the potential roles of the kiss signaling system in physiological processes, such as reproduction, nervous system activity and metabolism.

To verify the physiological function of *A. japonicus* kisspeptins, cultured oocytes were stimulated by different kisspeptins. As shown in [Figure 7C](#fig7){ref-type="fig"}, significant ERK phosphorylation signal can be detected by western blot assay in different kisspeptin-treated oocytes. This signal can be blocked by the kisspeptin antagonist pep234 (1.0 μM) in DrKiss1-10 or AjKiss1b-10 administrated cells (the inhibitory effect of pep234 was validated in vitro as shown in [Figure 7---figure supplement 3](#fig7s3){ref-type="fig"}). Further, detection by confocal microscopy of the p-ERK signal in treated oocytes demonstrated the activation of this pathway by AjKiss1b-10 and its inhibition by pep234 in *A. japonicus* cells ([Figure 7D](#fig7){ref-type="fig"}).

Having confirmed their functional activity in cultured oocytes, AjKiss1b-10 and pep234 were used to conduct further in vivo experiments. Sea cucumbers treated with AjKiss1b-10 for 40 days exhibited weight loss (p=0.0583, Tukey's multiple comparisons test, as shown in [Figure 7E](#fig7){ref-type="fig"}) and extremely significant intestinal degeneration (p=0.0001, Tukey's multiple comparisons test, as shown in [Figure 7F,G](#fig7){ref-type="fig"}), which are characteristic phenotypes of aestivating *A. japonicus* ([@bib62]). Moreover, extremely significant elevation of transcription of the gene encoding pyruvate kinase (PK) (p=0.0002, PBS vs. AjKiss1b-10, Tukey's multiple comparisons test, as shown in [Figure 7---figure supplement 4A](#fig7s4){ref-type="fig"}), which is the rate-limiting enzyme in the regulation of *A. japonicus* glycolysis ([@bib65]), was detected in the respiratory tree, whereas a significant decrease of PK transcription was found in the intestine (p=0.0188, PBS vs. AjKiss1b-10, Tukey's multiple comparisons test, as shown in [Figure 7---figure supplement 4A](#fig7s4){ref-type="fig"}). These data suggest that the *A. japonicus* kisspeptin system plays a role in the control of metabolic balance. To evaluate the potential role of AjKiss1b-10 in regulating reproductive activity, we examined the estradiol (E2) levels in the coelomic fluid of sea cucumber, but no significant difference was observed in animals treated with AjKiss1b-10 ([Figure 7---figure supplement 4B](#fig7s4){ref-type="fig"}).

The transcriptional expression of the *A. japonicus* kisspeptin precursor (*AjpreKiss*) and of the kisspeptin receptors (*AjKissR1/2*) was investigated at different stages of reproductive development using the qPCR method. Two-year-old sea cucumbers, with 85.29 ± 9.47 g body weight ([Figure 7---figure supplement 5A](#fig7s5){ref-type="fig"}), were collected and various tissues were sampled for further analysis. As shown in [Figure 7---figure supplement 5B](#fig7s5){ref-type="fig"}, notable changes in the relative gut mass and the relative ovary weight of the sea cucumbers were detected in the developing reproductive stage from November to April, the mature reproductive stage in May, after spawning in June, and during aestivation in August. At all stages, *AjKissR1/2* expression was detectable in the majority of sea cucumber tissues, especially after February ([Figure 7H](#fig7){ref-type="fig"}), whereas significant expression of *AjpreKiss* was found in the ANP from December to April with a peak value detected in February. Taken together, the high expression levels of *AjpreKiss* during reproductive development suggests its role in the regulation of seasonal reproduction, whereas the wide distribution of *AjKissR1* and *AjKissR2* in the other tissues investigated indicates diverse functions for these two receptors.

Discussion {#s3}
==========

The functional characterization of neuropeptides or secretory neurons of non-vertebrates contributes to our understanding of the evolutionary origin and conserved roles of the neurosecretory system in animals, especially in Ambulacrarians (deuterostomian invertebrates including hemichordates and echinoderms), which are closely related to chordates ([@bib58]; [@bib37]). The hypothalamic neuropeptide kisspeptin acts as a neurohormone and plays important roles in the regulation of diverse physiological processes in vertebrates, including reproductive development ([@bib41]; [@bib15]), metastasis suppression ([@bib9]), metabolism and development ([@bib53]; [@bib23]; [@bib24]), behavioral and emotional control ([@bib10]), and the innate immune response ([@bib20]).

A functional kisspeptin signaling system has been demonstrated in the chordate amphioxus ([@bib63]), and a number of invertebrate kisspeptin genes have been predicted recently ([@bib32]; [@bib14]; [@bib45]; [@bib46]; [@bib55]; [@bib8]), however, missing experimental identification of a kisspeptin-type system in non-chordates makes it difficult to determine whether this signaling system has an ancient evolutionary origin in invertebrates or whether it evolved de novo in the chordate/vertebrate lineages. In this study, two kisspeptin receptors from the sea cucumber *A. japonicus*, AjKissR1 and AjKissR2, have been identified as having a high affinity for synthetic kisspeptins from *A. japonicus* and vertebrates, and as sharing a similar G~αq~-dependent PLC/PKC signaling pathway with the mammalian kisspeptin signaling system. Results from the in vivo investigation indicate that the kisspeptin system in sea cucumber might be involved in the control of both metabolism and reproduction. Given the highly conserved intracellular signaling pathway and physiological functions revealed for the *A. japonicus* kisspeptin system, it is more likely that kisspeptin signaling might have originated from non-chordate invertebrates.

Two putative kisspeptin receptors can be activated by multiple synthetic kisspeptin-type peptides in *A. japonicus* {#s3-1}
-------------------------------------------------------------------------------------------------------------------

Kisspeptins or kisspeptin receptors in Chordata have been functionally recognized in various species. Virtual screening of the transcriptome and genome sequence data for neuropeptide precursors has made a great contribution to the prediction of kisspeptin and its receptor paralogous genes in Ambulacrarians and has provided valuable information for further investigation ([Figure 8](#fig8){ref-type="fig"}). In 2013, kisspeptin-type receptors were first annotated in the genome of the acorn worm *S. kowalevskii* and the purple sea urchin *S. purpuratus* ([@bib22]; [@bib32]). Moreover, a kisspeptin-type neuropeptide precursor with 149 amino-acid residues was identified in the starfish *Asterias rubens*, comprising two putative kisspeptin-type peptides, ArKiss1 and ArKiss2 ([@bib45]). Subsequently, in silico analysis of neural and gonadal transcriptomes enabled the virtual discovery of kisspeptins in the sea cucumbers *H. scabra* and *H. glaberrima* ([@bib55]). Moreover, the presence of kisspeptin-type peptides in extracts of radial nerve cords was confirmed by proteomic mass spectrometry in the crown-of-thorns starfish *A. planci* ([@bib52]). Recently, a 180-residue protein comprising two putative kisspeptin-type peptides has been predicted and a C-terminally amidated peptide GRQPNRNAHYRTLPF-NH~2~ was confirmed by mass spectrometric analysis of central nerve ring extracts of *A. japonicus* ([@bib8]). These advances provide a basis for experimental studies on the kisspeptin signaling system in echinoderms.

![Recently identified Kisspeptin or Kisspeptin receptor genes among some deuterostomes.\
The species, indicated by silhouette images downloaded from the *PhyloPic* database, were clustered in a phylogenetic tree and classified by different colors. Red highlighted 'R' indicates a whole-genome duplication event. Kiss/KissR indicates the identified Kisspeptin/Kisspeptin receptor gene, and KissL/KissRL indicates a predicted Kisspeptin-like/Kisspeptin-like receptor gene. Dashed symbols indicate pseudogenes. Arabic numerals indicate the number of genes identified or predicted from public data. The evolutionary tree of the indicated species was modified from [@bib40]. Image credits: all silhouettes from PhyloPic, human by T Michael Keeseyacorn; mouse by Anthony Caravaggi; platypus by Sarah Werning; duck by Sharon Wegner-Larsen; crocodile by B Kimmel; turtle by Roberto Díaz Sibaja; python by V Deepak; frog uncredited; coelacanth by Yan Wong; zebrafish by Jake Warner; spotted gar by Milton Tan; Branchiostoma by Mali\'o Kodis, photograph by Hans Hillewaert; acorn worm by Mali\'o Kodis, drawing by Manvir Singh; starfish by Hans Hillewaert and T Michael Keesey; sea cucumber by Lauren Sumner-Rooney; sea urchin by Jake Warner.](elife-53370-fig8){#fig8}

In the present study, we cloned the full-length of *Kiss* cDNA sequence from the ANP tissue samples, encoding a putative kisspeptin precursor, which has been predicted from the proteomic analysis of *A. japonicus* ([@bib8]) and synthesized the peptides AjKiss1a (32aa), AjKiss1a-15, AjKiss1a-13, AjKiss1a-10, AjKiss1b (18aa), and AjKiss1b-10, for further experimental characterization. Two candidate *A. japonicus* kisspeptin receptors were screened from genomic data and cloned from ovary tissue, on the basis of the sequence of the identified kisspeptin receptors ([@bib4]; [@bib13]; [@bib22]; [@bib32]; [@bib50]; [@bib17]; [@bib63]) and functionally characterized. Our data show that despite a low percentage homology between AjKissR1 and AjKissR2, both the receptors were efficiently activated by synthetic *A. japonicus* kisspeptin peptides (AjKiss1a and AjKiss1b), thereby triggering extensive Ca^2+^ mobilization and initiating significant receptor internalization, albeit with a different potency, when expressed in a mammalian cell line. This is consistent with previous studies demonstrating that in non-mammalian species, synthetic Kiss1 and Kiss2 activated kisspeptin receptors in vitro with differential ligand selectivity ([@bib38]; [@bib28]). In particular, the truncated peptide AjKiss1b-10 exhibited a high activity in eliciting intracellular Ca^2+^ mobilization in AjKissR1/2-expressing HEK293 cells, whereas the truncated peptides, AjKiss1a-15, AjKiss1a-13, and AjKiss1a-10, failed to activate the receptors. The functional activity of the truncated peptide AjKiss1b-10 is not unusual, considering that alternative cleavage occurs in the kisspeptin peptides of vertebrates ([@bib26]; [@bib28]); however, the inactivity of AjKiss1a-15, which has been identified from mass spectrometric detection in *A. japonicus* ([@bib8]), raises more questions about the functional and structural characteristics of this neuropeptide and requires further investigation.

Cross interaction of the kisspeptins and receptors between *A. japonicus* and vertebrates confirmed the existence of kisspeptin signaling systems in echinoderms {#s3-2}
----------------------------------------------------------------------------------------------------------------------------------------------------------------

In the mammalian genome, a single *Kiss1* gene produces a mature 54-amino-acid peptide, Kiss-54, which is further proteolytically truncated to 14- and 13-amino-acid carboxyl-terminal peptides, Kiss-14 and Kiss-13, with a common C-terminal decapeptide (Kiss-10) core ([@bib26]; [@bib39]). In non-mammalian vertebrates, two paralogous kisspeptin genes, *Kiss1* and *Kiss2*, are present in the genome of teleosts, producing two mature peptides that share the highly conserved Kiss-10 region with mammalian kisspeptin peptides ([@bib4]; [@bib28]; [@bib69]). Unlike mammalian and non-mammalian vertebrates, in the sea cucumber *A. japonicus*, only one kisspeptin gene was annotated and isolated. However, sequence analysis revealed that the kisspeptin gene encodes a 180-amino-acid peptide precursor, which is proteolytically cleaved to two mature peptides, consistent with other kisspeptins identified in the phylum Echinodermata ([@bib45]; [@bib46]; [@bib52]). Both putative mature peptides have a C-terminal Leu-Pro-Phe-amide motif, instead of the Arg-Phe-amide motif common in vertebrate kisspeptins, and exhibit a much lower identity with vertebrate kisspeptin sequences. Thus, the cross interaction of kisspeptin peptides and receptors between *A. japonicus* and vertebrates was further evaluated in this study.

Our specificity analysis showed that human, frog, and zebrafish kisspeptins, HsKiss1-10, XtKiss1b-10, and DrKiss1-10 and DrKiss2-10, were potent in activating both AjKissR1 and AjKissR2, whereas the human neuropeptide S (HsNPS, as a negative control) showed no potency to activate AjKissR1 or AjKissR2. Likewise, neuropeptides AjKiss1a and AjKiss1b could potentiate Ca^2+^ signaling by binding the human kisspeptin receptor HsKiss1R and the zebrafish kisspeptin receptors DrKiss1Ra/b, similar to the corresponding active decapeptides. This, to our knowledge, is the first experimental data directly confirming the connection between the kisspeptin systems of vertebrates and *A. japonicus*, therefore proving that this peptide system is present and active in non-chordate deuterostome species. Considering the high conservation of the neuropeptides in different echinoderms ([@bib46]; [@bib66]), our findings strongly suggest that the kisspeptin signaling system that exists in *A. japonicus* may be extend to other taxa in this phylum.

Conserved G~αq~/PLC/PKC/MAPK intracellular pathway mediated by the *A. japonicus* kisspeptin system provides insights into the evolution of kisspeptin signaling {#s3-3}
----------------------------------------------------------------------------------------------------------------------------------------------------------------

It is well established that in mammals, Kiss1R is a typical G~αq~-coupled receptor, triggering intracellular Ca^2+^ mobilization and the PLC/PKC signaling cascade in response to agonists ([@bib25]). However, accumulating evidence shows that in teleosts, although both kisspeptin receptors preferentially activate the G~αq~-dependent PKC pathway, one of them is also capable of triggering the G~αs~-dependent PKA cascade in response to kisspeptin challenge ([@bib38]; [@bib4]). Using CRE-Luc and SRE-Luc reporting assays, which help to discriminate between the AC/PKA and PLC/PKC signaling pathways, an amphioxus kisspeptin receptor was shown to trigger significant PKC but not PKA signaling when stimulated by two kisspeptin-type peptides using heterologous expression in cultured HEK293 cells ([@bib63]).

In this study, our data showed that, upon synthetic peptide stimulation, both AjKissR1 and AjKissR2 induced a rapid and transient rise in intracellular Ca^2+^ in a G~αq~ inhibitor FR900359-sensitive manner. Further functional characterization demonstrated that both AjKissR1 and AjKissR2 induced ERK1/2 activation via a G~αq~/PLC/PKC cascade. Although the G~αs~ protein has been implicated in the teleost kisspeptin receptors-mediated signaling pathway, we failed to collect distinct evidence to prove the involvement of G~αs~-dependent signaling in the *A. japonicus* kisspeptin system. Taken together, it is more likely that G~αq~-coupled signaling is highly conserved in the kisspeptin signaling systems from *A. japonicus* to mammals.

Reproductive and metabolic regulatory functions identified in *A. japonicus* revealed the ancient physiological roles of the kisspeptin system {#s3-4}
----------------------------------------------------------------------------------------------------------------------------------------------

Diverse physiological functions of the kisspeptin system have been reported in vertebrate species. In mammals, it is widely established that the kisspeptin signaling system is essential for HPG axis regulation, leading to reproductive control. Furthermore, the hypothalamic kisspeptin neurons have been found to stimulate pituitary gonadotropin-releasing hormone neurons, which express the kisspeptin receptor, providing a neural pathway for the mammalian kisspeptin neuronal system ([@bib36]). In non-mammalian species, especially in teleosts, the reproductive function of the kisspeptin system is still controversial in light of the normal reproductive phenotypes observed in fish in the absence of kisspeptins. A new theory has been proposed in which the nonreproductive functions beyond HPG regulation are the conserved roles of kisspeptins in vertebrates ([@bib56]; [@bib35]). Here, we applied multiple approaches to analyze the potential functions of the recently identified kisspeptin in *A. japonicus*, aiming to provide some insights into the ancient physiological roles of the kisspeptin system.

In the present study, the expressional distribution of the *A. japonicus* kisspeptin and its receptor proteins in multiple tissues suggests the involvement of the kisspeptin signaling system in the regulation of both reproductive and non-reproductive functions. The seasonal fluctuation of kisspeptin and receptors transcripts, consistent with the reproductive development process, indicates the possible functional involvement of this system in the control of seasonal reproduction in *A. japonicus*. Interestingly, the unequally expressed kisspeptin and receptor protein levels in gonads, comparatively high kisspeptin precursor level in testis, and high kisspeptin receptor protein levels in ovary tissue demonstrated in our study, suggests differential functions of the kisspeptin system in different genders of sea cucumber. Further investigation using both in vivo and in vitro experiments indicated a role for the kisspeptin signaling system in regulating metabolic balance and gut function in sea cucumber. Combining the feeding regulatory function of VP/OT-type neuropeptides characterized in echinoderm ([@bib37]) and the cross-talk between kisspeptin and VP/OT neural systems ([@bib19]; [@bib47]; [@bib35]), we suggest that a role of kisspeptin signaling in the regulation of the VP/OT system may exist in echinoderms, and the possible interaction between these two systems and an evolutionarily conserved function of the kisspeptin system is worthy of further exploration.

Materials and methods {#s4}
=====================

  -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  Reagent type\                   Designation                                                   Source or reference                                                                            Identifiers                                                  Additional information
  (species) or resource                                                                                                                                                                                                                                     
  ------------------------------- ------------------------------------------------------------- ---------------------------------------------------------------------------------------------- ------------------------------------------------------------ -------------------------------------------------------
  Cell line (*Homo sapiens*)      HEK293 cell line                                              The National Institutes of Health (Bethesda, MD)                                               RRID:[CVCL_0045](https://scicrunch.org/resolver/CVCL_0045)   Cell line maintained in this lab.

  Antibody                        Anti-phospho-ERK1/2(Thr^202^/Tyr^204^) (monoclonal, rabbit)   Cell Signaling Technology                                                                      CAT\#9101\                                                   IF (1:2000)
                                                                                                                                                                                               RRID:[AB_331646](https://scicrunch.org/resolver/AB_331646)   

  Antibody                        Anti-ERK1/2 antibody (monoclonal, rabbit)                     Cell Signaling Technology                                                                      CAT\#9102\                                                   IF (1:2000)
                                                                                                                                                                                               RRID:[AB_330744](https://scicrunch.org/resolver/AB_330744)   

  Antibody                        Anti-beta Tubulin (monoclonal, rabbit)                        Beyotime                                                                                       CAT\#AF1216                                                  IF (1:2000)

  Antibody                        FITC-conjugated goat anti-rabbit IgG (polyclonal, goat)       Beyotime                                                                                       CAT\#A0562                                                   IF (1:500)

  Antibody                        Cy3-conjugated goat anti-rabbit IgG (polyclonal, goat)        Beyotime                                                                                       CAT\#A0516                                                   IF (1:500)

  Antibody                        HRP-conjugated goat anti-rabbit IgG (polyclonal, goat)        Beyotime                                                                                       CAT\#A0208                                                   IF (1:500)

  Antibody                        anti-AjKiss1b-10 IgG (polyclonal, rabbit)                     ChinaPeptides                                                                                  CNE180821096                                                 Antigen sequence:\
                                                                                                                                                                                                                                                            CSRARPPLLPF-NH2\
                                                                                                                                                                                                                                                            IF (1:1000)

  Antibody                        anti-AjKissR1 Ser^150^\~Trp^174^IgG (polyclonal, rabbit)      Wuhan Transduction Bio                                                                         PC059                                                        Antigen sequence:\
                                                                                                                                                                                                                                                            SYTRYQFIIHPLKARAEWTSARVWW\
                                                                                                                                                                                                                                                            IF (1:1000)

  Sequence-based reagent          Primers for plasmid construction AjKissR1--EGFP               This paper                                                                                     PCR PRIMER                                                   FORWARD CGAATTCATGTTTGACGAAATGTTC EcoR I

  Sequence-based reagent          Primers for plasmid construction AjKissR1--EGFP               This paper                                                                                     PCR PRIMER                                                   REVERSE GTGGATCCCGAACGATACGATTCTGTTC BamH I

  Sequence-based reagent          Primers for plasmid construction FLAG--AjKissR1               This paper                                                                                     PCR PRIMER                                                   FORWARD GGAATTCATGTTTGACGAAATGTTC EcoR I

  Sequence-based reagent          Primers for plasmid construction FLAG--AjKissR1               This paper                                                                                     PCR PRIMER                                                   REVERSE CGGGATCCTCAAACGATACGATTCTGTTC BamH I

  Sequence-based reagent          Primers for plasmid construction AjKissR2--EGFP               This paper                                                                                     PCR PRIMER                                                   FORWARD CGAATTCATGGACAGCCTCTCAGC EcoR I

  Sequence-based reagent          Primers for plasmid construction AjKissR2--EGFP               This paper                                                                                     PCR PRIMER                                                   REVERSE CCGTCGACTGAGTTACAGTATTTGCTG SalI

  Sequence-based reagent          Primers for plasmid construction FLAG--AjKissR2               This paper                                                                                     PCR PRIMER                                                   FORWARD CCAAGCTTGGATGGACAGCCTCTCAGCGTT Hind III

  Sequence-based reagent          Primers for plasmid construction FLAG--AjKissR2               This paper                                                                                     PCR PRIMER                                                   REVERSE CGGGATCCCGTGAGTTACAGTATTTGCTGCAT Bam HI

  Sequence-based reagent          Primers for plasmid construction FLAG--HsKiss1R               This paper                                                                                     PCR PRIMER                                                   FORWARD CCAAGCTTGGATGCACACCGTGGCTAC Hind III

  Sequence-based reagent          Primers for plasmid construction FLAG--HsKiss1R               This paper                                                                                     PCR PRIMER                                                   REVERSE CGGGATCCTCAGAGAGGGGCGTTGTCCT Bam HI

  Sequence-based reagent          Primers for qPCR assays\                                      This paper                                                                                     PCR PRIMER                                                   FORWARDAGTGGACATCTGCAAGAGTATGG
                                  *AjKissR1*                                                                                                                                                                                                                

  Sequence-based reagent          Primers for qPCR assays\                                      This paper                                                                                     PCR PRIMER                                                   REVERSE CTTCCTGCGTAATGGTATCGGTA
                                  *AjKissR1*                                                                                                                                                                                                                

  Sequence-based reagent          Primers for qPCR assays\                                      This paper                                                                                     PCR PRIMER                                                   FORWARD TCTCGTTGTTGTCTTGACGTTTG
                                  *AjKissR2*                                                                                                                                                                                                                

  Sequence-based reagent          Primers for qPCR assays\                                      This paper                                                                                     PCR PRIMER                                                   REVERSETCGTCTGAAGTTTTCTCCCATGA
                                  *AjKissR2*                                                                                                                                                                                                                

  Sequence-based reagent          Primers for qPCR assays\                                      This paper                                                                                     PCR PRIMER                                                   FORWARD CCTACTGTCATTGCTCTGTGGAAC
                                  *AjpreKiss*                                                                                                                                                                                                               

  Sequence-based reagent          Primers for qPCR assays\                                      This paper                                                                                     PCR PRIMER                                                   REVERSECAAGGTCATCTTCGTCTTGTTCTC
                                  *AjpreKiss*                                                                                                                                                                                                               

  Sequence-based reagent          Primers for qPCR assays\                                      This paper                                                                                     PCR PRIMER                                                   FORWARD CACCACGTGGACTCAAAATG
                                  *β-*t*ubulin*                                                                                                                                                                                                             

  Sequence-based reagent          Primers for qPCR assays\                                      This paper                                                                                     PCR PRIMER                                                   REVERSE GAAAGCCTTACGACGGAACA
                                  *β-tubulin*                                                                                                                                                                                                               

  Sequence-based reagent          Primers for qPCR assays\                                      This paper                                                                                     PCR PRIMER                                                   FORWARD AAGGTTATGCTCTTCCTCACGC
                                  *β-actin*                                                                                                                                                                                                                 

  Sequence-based reagent          Primers for qPCR assays\                                      This paper                                                                                     PCR PRIMER                                                   REVERSE GATGTCACGGACGATTTCACG
                                  *β-actin*                                                                                                                                                                                                                 

  Recombinant DNA reagent         AjKissR1--EGFP                                                This paper                                                                                     Plasmid                                                      C-terminal EGFP-tag,\
                                                                                                                                                                                                                                                            backbone pEGFP-N1

  Recombinant DNA reagent         FLAG---AjKissR1                                               This paper                                                                                     Plasmid                                                      N-terminal FLAG-tag,\
                                                                                                                                                                                                                                                            backbone pCMV--FLAG

  Recombinant DNA reagent         AjKissR2--EGFP                                                This paper                                                                                     Plasmid                                                      C-terminal EGFP-tag,\
                                                                                                                                                                                                                                                            backbone pEGFP-N1

  Recombinant DNA reagent         FLAG--AjKissR2                                                This paper                                                                                     Plasmid                                                      N-terminal FLAG-tag,\
                                                                                                                                                                                                                                                            backbone pCMV--FLAG

  Recombinant DNA reagent         FLAG--HsKiss1R                                                This paper                                                                                     Plasmid                                                      N-terminal FLAG-tag,\
                                                                                                                                                                                                                                                            backbone pCMV--FLAG

  Recombinant DNA reagent         FLAG--DrKiss1Ra                                               This paper                                                                                     Plasmid                                                      N-terminal FLAG-tag,\
                                                                                                                                                                                                                                                            backbone pCMV--FLAG

  Recombinant DNA reagent         FLAG--DrKiss1Rb                                               This paper                                                                                     Plasmid                                                      N-terminal FLAG-tag,\
                                                                                                                                                                                                                                                            backbone pCMV--FLAG

  Recombinant DNA reagent         EGFP-tagged rat PKC isoforms (α, βI, βII and δ)               Kindly provided byDr Jin O-Uchi, University of Rochester and Dr Naoaki Sato, Kobe University   Plasmid                                                      C-terminal EGFP-tag,\
                                                                                                                                                                                                                                                            backbone pTB701

  Peptide, recombinant protein    AjKiss1a\                                                     This paper                                                                                                                                                  AGSLDc \< CLEASC \> EDVERRGRQPNRNAHYRTLPF-NH2
                                  (C-terminal amidated)                                                                                                                                                                                                     

  Peptide, recombinant protein    FITC--AjKiss1a (C-terminal amidated)                          This paper                                                                                                                                                  FITC--AGSLDc \< CLEASC \> EDVERRGRQPNRNAHYRTLPF-NH~2~

  Peptide, recombinant protein    AjKiss1a-15 (C-terminal amidated)                             This paper                                                                                                                                                  GRQPNRNAHYRTLPF-NH~2~

   Peptide, recombinant protein   AjKiss1a-10 (C-terminal amidated)                             This paper                                                                                                                                                  AHYRTLPF-NH~2~

  Peptide, recombinant protein    AjKiss1b\                                                     This paper                                                                                                                                                  SAVKNKNKSRARPPLLPF-NH~2~
                                  (C-terminal amidated)                                                                                                                                                                                                     

  Peptide, recombinant protein    AjKiss1b-10 (C-terminal amidated)                             This paper                                                                                                                                                  SRARPPLLPF-NH~2~

  Peptide, recombinant protein    HsKISS1-10 (C-terminal amidated)                              This paper                                                                                                                                                  YNWNSFGLRF-NH~2~

  Peptide, recombinant protein    XtKISS3/KISS1b-10 (C-terminal amidated)                       This paper                                                                                                                                                  YNVNSFGLRF-NH~2~

  Peptide, recombinant protein    DrKISS1-10 (C-terminal amidated)                              This paper                                                                                                                                                  YNLNSFGLRY-NH~2~

  Peptide, recombinant protein    DrKISS2-10 (C-terminal amidated)                              This paper                                                                                                                                                  FNYNPFGLRF-NH~2~

  Peptide, recombinant protein    pep234\                                                       This paper                                                                                                                                                  ac-(D-A)NWNGFG(D-W)RF-NH~2~
                                  (C-terminal amidated)                                                                                                                                                                                                     

  Commercial assay or kit         Rapid DNA Ligation kit                                        Beyotime                                                                                       CAT\#D7003                                                   

  Commercial assay or kit         SYBR PrimeScript RT reagent Kit                               TaKaRa                                                                                         CAT \#RR037A                                                 

  Commercial assay or kit         Iodine (125I) radioimmunoassay kit                            Beijing North Institute of Biotechnology                                                       S10940094                                                    

  Chemical compound, drug         Pertussis toxin (PTX)                                         Tocris Bioscience                                                                              Cat \#3097/50U                                               Specific inhibitor of G~αi~

  Chemical compound, drug         U73122                                                        Tocris Bioscience                                                                              Cat \#1268/10                                                PLC inhibitor

  Chemical compound, drug         BAPTA-AM                                                      Tocris Bioscience                                                                              Cat \#2787/25                                                Intracellular calcium chelator

  Chemical compound, drug         EGTA                                                          Tocris Bioscience                                                                              Cat \#2807/1G                                                Extracellular calcium chelator

  Chemical compound, drug         Gö6983                                                        Tocris Bioscience                                                                              Cat \#2285/1                                                 Broad spectrum PKC inhibitor

  Chemical compound, drug         FR900359                                                      Kindly provided by Dr Shihua Wu, Zhejiang University                                                                                                        Specific inhibitor of G~αq~
  -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Animal collection and treatment {#s4-1}
-------------------------------

For cDNA cloning and gene expression analysis in various tissues, individuals of the sea cucumber *A. japonicus* were collected from separate culture ponds in Qingdao (Shandong, China, in 2016--2017). Each batch was acclimated in seawater aquaria (salinity range: 32.21--34.13) for 7 days and further dissected, sampled, and stored in liquid nitrogen for future use or directly used for tissue culture. Individuals for in vivo experiments (94 ± 4.3 g) were collected from the same culture pond in November 2017, kept in a 500 L tank, and fed with a formulated diet (45% marine mud, 50% Sargasso, and 5% shrimp shell powder) before chemicals were administered. After 15 days, sea cucumbers were randomly assigned to different groups (10 individuals per group). AjKiss1b-10 was dissolved in PBS and intraperitoneal injection of 100 μL AjKiss1b-10 (concentration of 0.5 mg/mL diluted in PBS) or of PBS alone was conducted once every 2 days, at noon. After 40 days (10 December 2017 to 18 January 2018) of chemical administration, animals were dissected and the respiratory tree, intestine, muscle, and anterior part tissues were taken as samples from five individuals for each group, and stored in liquid nitrogen for future use. Coelom fluid was collected and stored at **−**20°C for E2 detection. This experiment was carried out on Xixuan Fishery Technology Island without temperature or light control (sea water temperature 11.5--7.0°C). Individuals used in the in vitro experiments (89 ± 2.4 g) were collected from the same culture pond in May 2017 and the respiratory tree, muscle, body wall, intestine, anterior part (containing the nerve ring), and ovary were dissected and further restored in **−**20°C for western blotting or washed with PBS three times, in aseptic conditions, for tissue culture and in vitro experiments.

Cell lines {#s4-2}
----------

HEK293 cells were provided from The National Institutes of Health (Bethesda, MD). Cells were authenticated by STR profiling and routinely tested for mycoplasma using commercial kits.

Cell culture and transfection {#s4-3}
-----------------------------

HEK293 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, HyClone) supplemented with 10% FBS (fetal bovine serum), 100 units/mL penicillin, 100 μg/mL streptomycin and 4.0 mM L-glutamine (Thermo Fisher Scientific) at 37°C in a humidified incubator containing 5% CO~2~. The plasmid constructs were transfected into HEK293 cells using X-tremeGENE HP (Roche), according to the manufacturer\'s instructions. Two days after transfection, stably expressing cells can be selected by the addition of 800 mg/L G418 to avoid impacts from inefficient transfection in experiments.

Bioinformatic searches and tools {#s4-4}
--------------------------------

The cDNA sequences were used to query known sequences in GenBank using the blastx utility, BLASTX 2.8.0+ (<http://blast.ncbi.nlm.nih.gov/>). The cDNA sequence of *A. japonicus* kisspeptin precursor or kisspeptin receptors was translated into the predicted amino-acid sequence with DNAMAN 8.0. Analysis of the physicochemical properties of proteins was based on Protparam (<http://www.expasy.org/tools/protparam.html>). Analysis of transmembrane regions in the protein was achieved by TMHMM (<http://topcons.cbr.su.se/>). The deduced amino-acid sequences were aligned using ClustalW. The color-align property was generated by the Sequence Manipulation Suite (<http://www.bioinformatics.org/sms2/color_align_prop.html>). Signal peptide was predicted by SignalP-5.0 Server (<http://www.cbs.dtu.dk/services/SignalP/>).

To construct the Maximum Likelihood (ML) phylogenetic tree, the Constraint-based Multiple Alignment Tool (<https://www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?CMD=Web>) was used to compute a multiple protein sequence alignment. The FastTree program (version 2.1) was then used to construct a tree based on the WAG and CAT model, which provided local support values that were based on the Shimodaira-Hasegawa (SH) test ([@bib42]). For the phylogenetic analysis of Kisses and outgroups, poorly aligned regions from the multiple alignment were automatically eliminated by trimAl (version 1.3, <http://trimal.cgenomics.org/>) using the automated1 option (Heuristic Method) ([@bib6]). According to the test of benchmark, the trimmed alignment always resulted in ML trees, which were of equal (36%) or significantly better (64%) quality than the tree produced by the complete alignment. The phylogenetic trees were visualized through the Evolview web server ([@bib54]).

Molecular cloning and plasmid construction {#s4-5}
------------------------------------------

To construct the AjKissR1/2 fusion expression plasmids, RT-PCR was performed using total RNA extracted from *A. japonicus* ovaries to synthesize template cDNA. PCR amplification of the coding sequences of *AjKissR1/2* was performed using specific primers, with restriction sites (see \'Key Resources Table\'). The corresponding PCR products were then cloned into pCMV--FLAG and pEGFP--N1 vectors using restriction enzymes and a Rapid DNA Ligation Kit (Beyotime, China). The FLAG--HsKiss1R plasmid was constructed using total synthesized DNA (Wuhan Transduction Bio) with specific primers containing restriction sites (see \'Key Resources Table\'). All constructs were sequenced to verify the correct sequences, orientations, and reading frames.

Intracellular calcium measurement {#s4-6}
---------------------------------

The fluorescent Ca^2+^ indicator Fura-2/AM was used to detect intracellular calcium flux ([@bib29]). Briefly, the HEK293 cells expressing the AjKissR1, AjKissR2 or vertebrate kisspeptin receptor were washed twice with PBS and suspended at 5 × 10^6^ cells/mL in Hanks' balanced salt solution. The cells were then loaded with 3.0 μM Fura-2/AM for 30 min and washed twice in Hanks' solution. They were then stimulated with the indicated concentrations of different predicted *A. japonicus* kisspeptins or vertebrate kisspeptins. Finally, intracellular calcium flux was measured for 60 s, by determining the ratio of excitation wavelengths at 340 and 380 nm using a fluorescence spectrometer (Infinite 200 PRO, Tecan, Männedorf, Switzerland). All of the experiments for measuring Ca^2+^ mobilization were repeated independently at least three times.

Receptor localization and translocation assay by confocal microscopy {#s4-7}
--------------------------------------------------------------------

For receptor-surface expression analysis, HEK293 cells expressing AjKissR1/2-EGFP were seeded onto glass coverslips in 12-well plates, coated with 0.1 mg/mL poly-L-lysine, and allowed to attach overnight under normal growth conditions ([@bib29]). The cells were washed three times with PBS and further stained with the membrane probe DiI (Beyotime) at 37°C for 5--10 min, fixed with 4% paraformaldehyde for 10 min, and then incubated with DAPI (Beyotime) for 5--10 min. For the translocation assays, the receptor-expressing cells were treated with 1.0 μM of various stimuli for 60 min, washed three times with PBS and then fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Finally, the cells were mounted in mounting reagent (DTT/PBS/glycerol, 1:8:2) and visualized by fluorescence microscopy on a Zeiss laser scanning confocal microscope, which was attached to a Zeiss Axiovert 200 microscope and linked to an LSM5 computer system.

PKC translocation assay by confocal microscopy {#s4-8}
----------------------------------------------

For the translocation analysis of various PKC subtypes, HEK293 cells co-transfected with FLAG--AjKissR1 or FLAG--AjKissR2 and various GFP-tagged rat PKC isoforms (α, βI, βII and δ, see \'Key Resources Table\') were seeded onto glass coverslips or six-well plates. After treatment with AjKiss1b-10 (1.0 μM) at 37°C for the indicated times, the cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Then, the cells were mounted in 50% glycerol and visualized by fluorescence microscopy using a Zeiss Axiovert 200 microscope linked to an LSM5 computer system. Excitation was performed at 488 nm, and the fluorescence detection used a 505--530 nm bandpass filter.

Antibodies {#s4-9}
----------

The primary antibodies used for p-ERK1/2, ERK1/2, or β-tubulin detection were: rabbit anti-phospho-ERK1/2(Thr^202^/Tyr^204^) antibody (1:2000; Cell Signaling Technology), rabbit anti-ERK1/2 antibody (1:2000; Cell Signaling Technology), and beta-tubulin rabbit monoclonal antibody (1:2000; Beyotime). To examine the *A. japonicus* kisspeptin precursor or AjKissR1 in various tissues of sea cucumber, AjKiss1b-10 or a peptide corresponding to amino acids Ser^150^\~Trp^174^ of AjKissR1, the second intracellular loop, was synthesized and injected into two rabbits, respectively. The polyclonal antibodies, rabbit anti-AjKiss1b-10 (1:1000) was prepared by ChinaPeptides, and anti-AjKissR1 (1:1000) was prepared by Wuhan Transduction Bio. The secondary antibodies used were HRP-conjugated goat anti-rabbit IgG, FITC-conjugated goat anti-rabbit IgG, and Cy3-conjugated goat anti-rabbit IgG (Beyotime).

Protein extraction and western blotting {#s4-10}
---------------------------------------

To examine the phosphorylation of ERK, cells that expressed AjKissr1/2 or other GPR54s were incubated for the indicated times with different concentrations of kisspeptins. Subsequently, cells were lysed with lysis buffer (Beyotime) that contained protease inhibitor (Roche) at 4°C for 30 min on a rocker and then scraped. Proteins were then electrophoresed on a 10% SDS polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% skimmed milk, then probed with rabbit anti-phospho-ERK1/2(Thr^202^/Tyr^204^) antibody (1:2000; Cell Signaling Technology), followed by detection using HRP-conjugated goat anti-rabbit IgG (Beyotime). Blots were stripped and reprobed using anti-ERK1/2 antibody (1:2000; Cell Signaling Technology) as a control for protein loading.

To detect AjKissR1 in different tissues of sea cucumber, the respiratory tree, intestine, muscle, nerve ring, and ovary were sampled and homogenized with lysis buffer (Beyotime) that contained protease inhibitor (Roche) at 4°C. Comparable concentrations of proteins were then electrophoresed on a 10% SDS polyacrylamide gel and transferred to PVDF membranes. Membranes were blocked with 5% skimmed milk, then probed with rabbit anti-AjKissR1 serum (1:1000), followed by detection using HRP-conjugated goat anti-rabbit IgG (Beyotime). Samples were probed in parallel with anti-tubulin antibody (Beyotime) as control for protein loading.

To detect AjpreKiss or its mature peptide in different tissues of sea cucumber, tissues were sampled and treated following the same protocol for receptor detection. Comparable concentrations of proteins were then electrophoresed on a 15% SDS polyacrylamide gel (preparation of 20 mL gel solution: 4.6 mL dH~2~O, 10.0 mL 30% polyacrylamide solution, 5.0 mL 5 mol/L Tris \[pH8.8\], 0.2 mL 10% SDS solution, 0.2 mL 10% ammonium persulphate solution, 8.0 μL TEMED) and transferred to PVDF membranes (180 mA, 40 min for Kiss detection and 180 mA, 70 min for tubulin detection). Membranes were blocked with 5% skimmed milk, then probed with rabbit anti-AjKissR1 serum (1:1000), followed by detection using HRP-conjugated goat anti-rabbit IgG (Beyotime). Samples were probed in parallel with anti-tubulin antibody (Beyotime) as a control for protein loading.

Immunoreactive bands were detected with an enhanced chemiluminescent substrate (Beyotime), and the membrane was scanned using a Tanon 5200 Chemiluminescent Imaging System (Tanon Science and Technology, Shanghai, China).

Ligand competition binding assay {#s4-11}
--------------------------------

A fluorescence-activated cell sorter (FACS) was used to detect the binding ability of kisspeptins with AjKissR1 or AjKissR2. HEK293 cells expressing FLAG--AjKissR1 or FLAG--AjKissR2 were washed with PBS that contained 0.2% bovine serum albumin (FACS buffer). We designed and synthesized an N-terminal FITC-labeled AjKiss1a peptide (see \'Key resources table\'). Different kisspeptins were diluted in the FACS buffer to different concentrations, then added to cells that were incubated on ice for 60--90 min. Cells were washed thrice with the FACS buffer and re-suspended in the FACS buffer with 1% paraformaldehyde for 15 min. The binding activity of the indicated kisspeptin peptides with AjKissR1 or AjKissR2 was determined by measuring the fluorescence of FITC and was presented as a percentage of total binding.

Immunofluorescence assay on paraffin-embedded tissue sections {#s4-12}
-------------------------------------------------------------

Paraffin sections were baked at 60°C for 2--4 hr and placed in xylene for 15 min, twice. The slides were washed twice in 100% ethanol for 10 min each, then in 95% ethanol for 10 min, 85% ethanol for 5 min, 70% ethanol for 5 min, and 50% ethanol for 5 min, followed by washing with dH~2~O for 5 min, and finally washing with PBS for 5 min. Antigen unmasking was performed in sodium citrate buffer (pH 6) for 10 min at 97°C, and then cooled to room temperature. Endogenous peroxidases were blocked by 10 min incubation in 3.0% hydrogen peroxide. Nonspecific antigens were blocked by a 60 min incubation in 0.3% bovine serum albumin (BSA) in TBST. Slides were incubated with primary antibodies overnight after removing the blocking solution, followed by 2 hr incubation with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (FITC-labeled goat anti-rabbit IgG (H+L), Beyotime). Slides were washed with dH~2~O, mounted with antifade mounting medium (Beyotime), and imaged by confocal microscopy.

Real-time quantitative PCR (qRT-PCR) {#s4-13}
------------------------------------

For qRT-PCR, *β-actin* (ACTB) and *β-tubulin* (TUBB) were chosen as the internal control (housekeeping) genes and gene-specific primers were designed based on the ORF sequences ([@bib65]; [@bib68]). Specific qRT-PCR primers for *AjKissR1/2* and *AjKiss1* were designed based on CDS (see \'Key resources table\'). The primers were tested to ensure the amplification of single discrete bands, with no primer-dimers. qRT-PCR assays were carried out using the SYBR PrimeScript RT reagent Kit (TaKaRa, Kusatsu, Japan) following the manufacturer's instructions and using ABI 7500 Software v2.0.6 (Applied Biosystems, UK). The relative level of gene expression was calculated using the 2^--△Ct^ method and data were normalized by geometric averaging of the internal control genes ([@bib30]; [@bib61]).

Tissue culture and treatment {#s4-14}
----------------------------

For in vitro experiments, the ovary and respiratory tree tissues were cut into small pieces of approximately 1 mm^3^ and cultured in Leibovitz L-15 medium (HyClone) supplemented with 12.0 g/L NaCl, 0.32 g/L KCl, 0.36 g/L CaCl~2~, 0.6 g/L Na~2~SO~4~, 2.4 g/L MgCl~2~, 0.6 g/L glucose at 18°C in a humidified incubator ([@bib64]). For ERK1/2 phosphorylation or immunofluorescence assays, samples were evaluated or fixed after 2 hr of ligand/PBS administration, with a 4 hr pre-treatment of pep234/PBS.

Radioimmunoassay {#s4-15}
----------------

Levels of estradiol (E2) in coelomic fluid or culture medium were measured using the Iodine (^125^I) method ([@bib31]). In brief, estradiol levels were measured using Iodine (^125^I) radioimmunoassay kits (Beijing North Institute of Biotechnology, Beijing, China), according to the manufacturer's protocol. The binding rate is highly specific with an extremely low cross-activity to other naturally occurring steroids, which was less than 0.1% to most circulating steroids.

Data statistics {#s4-16}
---------------

Statistical analysis was done with GraphPad Prism (version 7.0). Statistical significance was determined using one-way ANOVA followed by Tukey's multiple comparisons test. Probability values that were less than or equal to 0.05 were considered significant, and less than or equal to 0.001 were considered extremely significant (\*p≤0.05, \*\*p≤0.01), and P values were indicated in the legend of the figures. All error bars represent the standard error of the mean (SEM), and all experimental data were gathered from at least three independent experiments showing similar results.

Funding Information
===================

This paper was supported by the following grants:

-   http://dx.doi.org/10.13039/501100001809National Science Foundation of China 41876154 to Tianming Wang.

-   http://dx.doi.org/10.13039/501100001809National Science Foundation of China 41406137 to Tianming Wang.

-   http://dx.doi.org/10.13039/501100001809National Science Foundation of China 41606150 to Jingwen Yang.

-   Center for Ocean Mega-Research of Science, Chinese Acadamy of Science COMS2019Q15 to Tianming Wang.

The authors of this paper would like to thank Prof. Igor Yu Dolmatov from the National Scientific Center of Marine Biology---Russian Academy of Sciences for his assistance on histomorphological analysis, Dr Xiaoshang Ru from Institute of Oceanology, Chinese Academy of Sciences for his assistance on sea cucumber sampling and suggestion on the discussion, Prof. Dongdong Xu for his technical assistance and equipment usage, and Dexiang Huang from Wang's lab for his technical assistance on phylogenetic analyses. This work was supported by the National Science Foundation of China (Nos. 41876154, 41406137 and 41606150) and Key Deployment Project of Center for Ocean Mega-Research of Science, Chinese Academy of Science (COMS2019Q15).

Additional information {#s5}
======================

No competing interests declared.

Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Project administration.

Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology.

Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology.

Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Project administration.

Validation, Investigation, Methodology.

Investigation.

Investigation.

Investigation.

Investigation.

Investigation.

Investigation.

Investigation.

Formal analysis, Methodology.

Funding acquisition, Investigation, Methodology.

Conceptualization, Supervision, Validation, Project administration.

Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Project administration.

Additional files {#s6}
================

Data availability {#s7}
=================

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, 2, 3, 4, 5 and 7.

The following datasets were generated:

ZhangXSunLYuanJSunYGaoYZhangLLiSDaiHHamelJ-FLiucYuYLiuSLinWGuoKJinSXuPStoreyKBHuanPZhangTZhouYZhangJLinCLiXXingLHuoDSunMWangLMercierALiFYanghXiangJ2017The sea cucumber genome provides insights into morphological evolution and visceral regenerationNCBI BioProjectPRJNA354676

TianmingW2019Apostichopus japonicus kisspeptin receptor (Kissr1) mRNA, complete cdsNCBI GenBankMH709114

WangT2019Apostichopus japonicus kisspeptin receptor (Kissr2) mRNA, complete cdsNCBI GenBankMH709115

10.7554/eLife.53370.sa1

Decision letter

Tessmar-Raible

Kristin

Reviewing Editor

University of Vienna

Austria

Arnone

Maria Ina

Reviewer

Stazione Zoologica Anton Dohrn

Italy

Terblanche

Vera

Reviewer

Germany

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

**Acceptance summary:**

Your work is an impressive analysis of the kisspeptin signaling system ranging from the in silico identification, receptor deorphanization to functional tests in the sea cucumber. This is not only interesting in the context of the evolution of neuropeptidergic systems in bilaterians in general, but particularly also in understanding possible evolutionary ancient mechanisms underlying animal seasonality.

**Decision letter after peer review:**

Thank you for submitting your article \"Existence and functions of hypothalamic kisspeptin neuropeptide signaling system in a non-chordate deuterostome species\" for consideration by *eLife*. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Diethard Tautz as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Maria Ina Arnone (Reviewer \#2); Vera Terblanche (Reviewer \#3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

Wang et al. performed a detailed study of the echinoderm Kisspeptin/receptor system, using the sea cucumber *Apostichopus japonicus* as a model. They identified 2 preprohormones and three receptors, of which they study both peptide hormones and two receptors in detail. They performed phylogenetic analyses, receptor activation and signaling studies, Western blot analyses and immunohistochemistry. The authors also provide an initial characterization of this peptidergic system in *Apostichopus japonicus* physiology. Based on those studies they come to important conclusions about the evolutionary conservation of the kisspeptin system.

Their findings are of potential great interest, being the first demonstration of functioning of this system outside vertebrates, with important implications for the understanding of the evolution of vertebrate (neuro)secretory systems, as well as seasonal regulation of reproduction and physiology.

However, the manuscript requires several essential improvements especially in its presentation (details below).

Essential revisions:

1\) The technical information necessary to evaluate the manuscript is either missing or spread across various places. This includes all primary metadata, sequences of synthetic peptides used for receptor, physiological and antibody studies. Metadata are entirely missing. In case of the peptides, it is not specified in the main text whether the synthetic Kiss peptides used in the experiment are amidated. This could only be found when digging through the supplementary tables.

2\) Connected to the missing technical information, there is insufficient information on the replicates used in the different studies. For instance, regarding the Ca^2+^ mobilization experiments (Figure 2B1 and B2). How representative are these experiments? How many times were they repeated and how reproducible were the results? In other cases a number \"n\" for replicates is mentioned, but it is unclear how the replicates (e.g., technical, biological) were defined.

3\) The phylogenetic trees require major improvements. De-code accession numbers in trees for species to make it better understandable to the reader without having to consult the databases for every branch. This is essential to see what the tree is really showing (and how representative it is.)

The critical bootstrap value for the Kisspeptin family preprohormone group is only 34 -- this means there is no real support for this group as a common branch and hence, in a strict sense, this would argue against a common evolutionary ancestry. On the other hand- such seemingly unsupported grouping is not totally uncommon for preprohormones across larger phylogenetic distances (e.g. due to the relative shortness of the peptide chains that can be used for the comparison), but then the authors need to discuss this issue and provide other reasons (such as their receptor homology, deorphanization and cross-binding studies) to argue their case for a preprohormone group that derived from a common evolutionary ancestor. Genomic gene structure information on the pre-prohormone could provide additional support (e.g. exon/intron boundaries; microsyntheny).

4\) Figure 5D, E: The data on EGFP-PKC isoform recruitment to the plasma membrane assays shown in Figure 5D are not convincing. In some cases the potential membrane signal is largely obscured by the high cytoplasmic expression. Is there a way to quantify this using a histogram? Maybe this figure is also not strictly necessary to support the findings.

5\) The specificity of the antibodies raised against AjKiss1b-10 and AjKISS-R1 is of major concerns.

There should be experiments showing that the antibody recognizes specifically the antigen. For protein blots, this could be achieved by probing lysates of non-transfected and transfected mammalian neuroendocrine cells, which could process the AjKiss precursor. The same goes for immunofluorescence, e.g. compare transfected and plasmid-only transfected tissue culture cells. In AjKISS-R1 transfected cells does the antibody signal co-localize with the GFP signal to the membrane?

Protein blots:

What kind of gel did the authors use for this experiment in Figure 6A? AjKiss1b-10 is 10 amino acid long (\~ 1.1 kDa). Since peptides this small are unlikely to be retained after transfer to PVDF, the authors should specify the experimental procedure to obtain this blot. Molecular weight markers would be useful to confirm the signals from the Aj tissues sampled are from the unprocessed or the partially processed precursor or the mature peptide. Was the anti-AjKiss1b-10 raised against the amidated peptide? If so, why the signals from the mature peptide are not detected (\"Western Blot analysis of *A. japonicus* kisspeptin precursor\")? Why is Kisspeptin detected in OVA by western and not by immunofluorescence?

6\) There are further major omissions of explanations throughout the text, e.g.:

In Figure 4A, the authors completely omit to discuss the PTX treatment shown in the panels. Does PTX stand for Pertussis toxin? The authors need to mention this treatment in the text and explain the reason to use PTX.

Explain AjKiss1b-10 in the text. Same for in the Figure 4 legend. Please consider that not all readers are familiar with the literature on Kisspeptins or neuropeptides in general. Also, please, pick one naming system and keep using it; i.e., use either Kp or Kiss or Kisspeptin. Same for receptor naming.

Subsection "In silico identification of Kps and Kp receptors", first paragraph: is that the same sequence identified by Swansa-ard et al./Chen et al? If yes state that and those lines can be omitted as it also repeats the Introduction.

Figure 5A1. What time (5 min, 10 min, etc. stimulation) the concentration-dependence blots refer to?

Figure 5A2 (bar graph). Why isn\'t the strongest signal for AjKissR2 set at 100% as in the case of AjKissR1?

Figure 6D. Upper left panel. What treatment is this?

Figure 6F. Explain what tissue index measures.

Figure 7: Why does this figure indicate presence of only one kisspeptin receptor in sea cucumber if the presented study finds two?

Substantial editing work is needed to allow readers to more easily follow the data. Generally, the figure legends and the Materials and methods section need the most attention.

Figures. Make larger panels, please. Some of the labels on the panels were very difficult, if not impossible, to decipher (for instance, 1C, 1E, 5A, 5B, 5C). Check for typos in the figure labels (for instance, Figure 2B: 1 μM instead of 1 um).

Materials and methods.

Animals should not be listed as \"Materials\". Transient and stable HEK293 cells expressing AjKissRs were generated, but it is not explained why. A protocol for DiI staining (Figure 2A) is not provided.

Receptor internalization (shown in Figure 2C) mentions 30 min, but the legend says 60 min. In the same experiments, it is said that cells were incubated with DAPI for several minutes before translocation. DAPI is not a vital stain; did the authors mean Hoechst? Species name: sometimes *A. japonicas*, at others it is *A. japonicus.*

Subsection "Data statistics": please refer to the exact experiments for which these statistic methods were used.

7\) In general, a conclusive sentence about what each of the experiments shows would greatly help the reader, e.g.- Subsection "Physiological functions of the Kp signaling system in *A. japonicus*", third paragraph: a one line conclusion on kiss signalling functions inferred from the effects of persistent kiss1a/1b treatment would be nice.

Do you think it maintains metabolic balance, or does it lead to the mobilisation of lipids from the fat tissues?

8\) The data on the potential function in seasonal control of reproduction are interesting, but as presented at present rather confusing, as they seem to be partly somewhat contradictory.

The statements in the second and last paragraphs of the subsection "Physiological functions of the Kp signaling system in *A. japonicus*, would suggest an important role in regulating the maturation of the germline, especially oocytes before/during the peak season of reproduction. How does this fit with the findings described in the third paragraph of the aforementioned subsection, describing what rather seems to be a role in aestivation?

9\) Final conclusion: The statement that it \"originally evolved in this hypothalamic neuropeptide system\" implies that it evolved along with the hypothalamus in higher vertebrates. The work presented by the authors does not support this claim, but rather implies that the molecular system itself (including the use of Gaq-PKC cascade in Kp signalling) is likely an ancestral state and is now used by the vertebrate hypothalamus. To draw evolutionary conclusion about an ancestral hypothalamic system the study would have to include other markers for vertebrate hypothalamic cell types (e.g. nkx2.1) and compare how kisspeptin localizes relative to the expression of these other markers. Furthermore, some places of kisspeptin detection- OVA, Tes, ANP- don\'t make sense with an \"ancestral neurosecretory system\" in the sense of an ancestral tissue unit. The authors should revise their text, heading and conclusions according to this, rather pointing out the molecular conservation, as well as the possibly interesting conserved link in the regulation of seasonal reproduction.

\[Editors\' note: further revisions were suggested prior to acceptance, as described below.\]

Thank you for resubmitting your work entitled \"Existence and functions of hypothalamic kisspeptin neuropeptide signaling system in a non-chordate deuterostome species\" for further consideration by *eLife*. Your revised article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Diethard Tautz as the Senior Editor.

The manuscript has been significantly improved by the additional experiments and clarifications, but there are some remaining issues that need to be addressed before acceptance, as outlined below. All aspects can be addressed by text or figure amendments. There are no further experiments needed.

1\) At present there is no strong evolutionary evidence for this system to homologous to the hypothalamus. We would thus ask to remove the word \"hypothalamic\" from the title.

Title suggestion:

\"Existence and functions of a kisspeptin neuropeptide signaling system in a non-chordate deuterostome species\"

2\) \"Through the evaluation of Ca^2+^ mobilization and other intracellular signals, we found that *A. japonicus* kisspeptins dramatically activated two receptors (AjKissR1 and AjKissR2), via a GPCR-mediated Gαq/PLC/PKC/MAPK signaling pathway, that have functions corresponding to those of the vertebrate kisspeptin system\". There is something wrong with this statement. Again, the GPCR-mediated Gαq/PLC/PKC/MAPK signaling pathway was shown in a mammalian cell line surrogate. So, while ex vivo experiments show ERK (i.e., MAPK cascade) involvement (Figure 7C and D) the authors have not shown that in/ex vivo Aj kisspeptin signaling involves PLC and PKC.

We would thus suggest to rephrase this in the following way (or something similar):

\"Through the evaluation of Ca^2+^ mobilization and other intracellular signals, we found that *A. japonicus* kisspeptins activate two receptors (AjKissR1 and AjKissR2) via a GPCR-mediated Gαq/PLC/PKC/MAPK signaling pathway in a mammalian cell line. Albeit likely it remains to be shown if the same signaling cascade also occurs in vivo in its seemingly conserved function in reproductive control.\"

3\) Subsection "In silico identification of kisspeptins and kisspeptin receptors". Change \"ANP, containing nerve ring\" to \"ANP, containing the nerve ring\".

4\) Subsection "In silico identification of kisspeptins and kisspeptin receptors". Change \"frames\" to \"frame\".

5\) Figure 3B legend. Change \"3-B\" to \"3B\".

6\) Figure 4 legend. It recalls the Figure 4 source data. Here, there is a mislabelling, as the linked excel files has individual worksheet labelled as 3A, 3B etc. -- they should be 4A, 4B etc, instead. The authors should check all their supplementary files for correct labelling.

7\) \"inhibitory effect of pep234 was preapproved in vitro\", should be changed to \"inhibitory effect of pep234 was validated in vitro\".

8\) \"Further detection of the pERK signal in AjKiss1b-10 treated oocytes by confocal microscopy demonstrated the physiological activation of this pathway by AjKiss1b-10 and pep234 on *A. japonicus* cells\" should be changed to \"Further, detection by confocal microscopy of the p-ERK signal in treated oocytes demonstrated activation of this pathway by AjKiss1b-10 and its inhibition by pep234 in *A. japonicus* cells\".

9\) \"Samples were collected and fixed after 2 h of ligand administration with or without a 4 h pre-treatment of pep234, in optimized L15 medium at 18 °C\". Should be changed to \"Samples were collected and fixed after 2 h of ligand administration with or without a 4 h pre-treatment with pep234, in optimized L15 medium at 18 °C\". Also, is this the same treatment for the samples shown in Figure 7C? If so, it should be clearly stated in the legend for panel 7C.

10\) After \"different potency\", we suggest the addition of \", when expressed in a mammalian cell line\".

11\) Change statement from \"therefore proving the functionality of this neuropeptide system in non-chordate species\" to \"therefore proving that this peptide system is present and active in non-chordate deuterostome species\".

12\) \"an amphioxus kisspeptin receptor was shown to trigger significant PKC and not PKA signaling, when stimulated by two kisspeptin-type peptides (Wang et al., 2017).\" Please, make clear that these experiments too were done using heterologous expression in HEK293 cells.

13\) It is stated that \"AjKissR1 and AjKissR2 induced ERK1/2 activation via a Gαq/PLC/PKC cascade. Our results showed that no significant accumulation of cAMP was detected in response to agonist treatment\". We could not find any experiment in the manuscript measuring cAMP accumulation. The authors should amend the text accordingly.

14\) All images of the Western blots are over-contrasted. This needs to be corrected and size markers also added to Figure 7 (not just the figure supplement).

15\) Carefully check for missing and correct scalebars. Figure 3: According to the scale bar these HEK cells are huge. Sure the scale bar is correct? Figure 6D, E: scale bars are entirely missing.

16\) There is still some remaining concern about the specificity of the antibody against AjKiss1b. We find it pretty unusual that an antibody that was generated against an amidated peptide would now only rather recognize the precursor (or if we played devil\'s advocate- a much larger band in the Western that could have nothing to do with the actual peptide). We would thus ask the authors to insert a cautionary sentence in the text.

10.7554/eLife.53370.sa2

Author response

> Essential revisions:
>
> 1\) The technical information necessary to evaluate the manuscript is either missing or spread across various places. This includes all primary metadata, sequences of synthetic peptides used for receptor, physiological and antibody studies. Metadata are entirely missing. In case of the peptides, it is not specified in the main text whether the synthetic Kiss peptides used in the experiment are amidated. This could only be found when digging through the supplementary tables.

As the reviewers suggested, in the revised manuscript, we have added the primary metadata in xlsx or txt format linked with the figures in main text or the supplementary figures, and the Key Resources Table is now incorporated within the main text at the very beginning of the Materials and methods section to provide the detailed information about the sequences and amidation of indicated synthetic peptides, as well as the characters of antibodies, especially for the customized polyclonal antibody for AjKiss1b-10 and AjKissR1. We thank the reviewers for their suggestion.

> 2\) Connected to the missing technical information, there is insufficient information on the replicates used in the different studies. For instance, regarding the Ca^2+^ mobilization experiments (Figure 2B1 and B2). How representative are these experiments? How many times were they repeated and how reproducible were the results? In other cases a number \"n\" for replicates is mentioned, but it is unclear how the replicates (e.g., technical, biological) were defined.

We have now provided the information on the replicates used in different experiments, which is now introduced in every figure legend and the "n" for animals or sample numbers is also clearly denoted in the revised manuscript as the reviewers suggested, and we thank the reviewers for this suggestion.

> 3\) The phylogenetic trees require major improvements. De-code accession numbers in trees for species to make it better understandable to the reader without having to consult the databases for every branch. This is essential to see what the tree is really showing (and how representative it is.)
>
> The critical bootstrap value for the Kisspeptin family preprohormone group is only 34 -- this means there is no real support for this group as a common branch and hence, in a strict sense, this would argue against a common evolutionary ancestry. On the other hand- such seemingly unsupported grouping is not totally uncommon for preprohormones across larger phylogenetic distances (e.g. due to the relative shortness of the peptide chains that can be used for the comparison), but then the authors need to discuss this issue and provide other reasons (such as their receptor homology, deorphanization and cross-bindind studies) to argue their case for a preprohormone group that derived from a common evolutionary ancestor. Genomic gene structure information on the pre-prohormone could provide additional support (e.g. exon/intron boundaries; microsyntheny).

We thank the reviewers for pointing out these issues. The abbreviated Latin names of species and protein names are now represented in trees, instead of accession numbers, to make it better understandable to the readers. To get more representative trees for preprohormone groups, a new strategy for ML tree construction is now applied in kisspeptin and kisspeptin receptor phylogenetic analyses. As introduced in the Materials and methods section of the revised text, our new approach mainly includes using Constraint-based Multiple Alignment Tool (https://www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?CMD=Web) for the multiple protein sequence alignment, and the FastTree program (version 2.1) for construction of the trees based on WAG and CAT model, and providing local support values based on the Shimodaira-Hasegawa (SH) test. For the phylogenetic tree of peptides, the poorly aligned regions from the multiple sequence alignment are automatically removed by trimAl. The trees were then visualized through Evolview web server. The revised phylogenetic tree for kisspeptin analysis represents much higher local support value for topological stability of the tree. Though, in the current tree, the AjKiss is clustered with predicated kisspeptin from other two sea cucumbers and then grouped with chordate kisspeptins under the high local support value of 81%, we still believe that the bioinformatical analysis can't support the definition of this screened Kisspeptin-like gene. We do agree with reviewers that gene structure information on the pre-prohormones could provide additional support and now it has been provided in the revised Figure 1. The conserved gene structure is displayed from comparative analysis of sea cucumber, zebrafish and human *preKiss* genes. We believe that these data, combined with the receptor homology, deorphanization and cross-binding results from our studies, should be sufficiently convincing to demonstrate the evolutionary homology of AjKiss and chordate kisspeptins.

> 4\) Figure 5D, E: The data on EGFP-PKC isoform recruitment to the plasma membrane assays shown in Figure 5D are not convincing. In some cases the potential membrane signal is largely obscured by the high cytoplasmic expression. Is there a way to quantify this using a histogram? Maybe this figure is also not strictly necessary to support the findings.

As the reviewers pointed out, we have performed additional experiments to address the concern on PKC isoform recruitment to the plasma membrane assays using plasmids of GFP-tagged rat PKC isoforms (α, βI, βII and δ) (details are shown in Key Resources Table) and obtained much more convincing data. As shown in the now revised Figure 6D and E, upon activation by AjKiss^-1^b, PKCα, βI and βII were clearly translocated to the plasma membrane, but not PKCδ. These new data suggest that PKCα, βI and βII are involved in the AjKissR1 and R2-mediated downstream signaling pathways.

> 5\) The specificity of the antibodies raised against AjKiss1b-10 and AjKISS-R1 is of major concerns.
>
> There should be experiments showing that the antibody recognizes specifically the antigen. For protein blots, this could be achieved by probing lysates of non-transfected and transfected mammalian neuroendocrine cells, which could process the AjKiss precursor. The same goes for immunofluorescence, e.g. compare transfected and plasmid-only transfected tissue culture cells. In AjKISS-R1 transfected cells does the antibody signal co-localize with the GFP signal to the membrane?

Detection of kisspeptin in non-transfected vs. transfected mammalian neuroendocrine cells should be a good approach for the analysis of intracellular processing and for assessing specificity of the antibody. We tried to develop this expression system using transfected mammalian neuroendocrine cells but failed due to their slow growth hence very difficult to culture. In addition, we were not allowed to enter the laboratory until now due to the coronavirus outbreak.

Therefore, to provide conclusive evidence of the antibody specificity raised against AjKiss1b-10, instead, we have collected fresh tissue samples from the sea cucumber for western blotting. As shown in Figure 7---figure supplement 1A, one band of AjKiss1b-1 (or AjKiss1b) and another about 20 KDa band can be detected. This 20 KDa band in tissue samples should be *Apostichopus japinicus* Kisspeptin precursor (theoretically deduced molecular weight of AjpreKiss is 20.29 KDa).

For assessing the specificity of anti- AjKissR1 anti-body, we performed western blot and immunofluorescence assays in HEK293 cells. As shown in Figure 7---figure supplement 1B and C, using anti- AjKissR1 antibody by western blot, we detected a 65 KDa band of AjKissR1-EGFP (theoretically deduced molecular weight 68 KDa) from HEK 293 cells; we also showed a 45 KDa band from the sea cucumber ovarian sample (theoretically deduced molecular weight 43.46 KDa). Using the immunofluorescence assay, co-localization of Cy3 and GFP in AjKissR1-expressing cells, but not in AjKissR2-expressing cells, was observed by confocal microscopy. Collectively, these data have demonstrated that the antibodies raised against AjKiss1b-10 and AjKISS-R1 are specifically recognizing the targeted proteins.

> Protein blots:
>
> What kind of gel did the authors use for this experiment in Figure 6A? AjKiss1b-10 is 10 amino acid long (\~ 1.1 kDa). Since peptides this small are unlikely to be retained after transfer to PVDF, the authors should specify the experimental procedure to obtain this blot. Molecular weight markers would be useful to confirm the signals from the Aj tissues sampled are from the unprocessed or the partially processed precursor or the mature peptide. Was the anti-AjKiss1b-10 raised against the amidated peptide? If so, why the signals from the mature peptide are not detected (\"Western Blot analysis of *A. japonicus* kisspeptin precursor\")? Why is Kisspeptin detected in OVA by western and not by immunofluorescence?

To detect kisspeptin precursor or its mature peptide in different tissues of sea cucumber, protein samples were electrophoresed on a 15% SDS polyacrylamide gel (details are provided in the Materials and methods section of revised text) and transferred to PVDF membranes (180 mA, 40 min for kisspeptin detection). Molecular weight markers imaged in bright field are now provided to show the size of the bands (as shown in Figure 7---figure supplement 1). The results indicate that the detected signal mainly shows the unprocessed precursor.

The anti-AjKiss1b-10 antibody was raised against the amidated peptide, but the signals from the mature peptide are not detected, because the concentration of the mature peptide in tissue samples is too low to be detected; it may also be possible that this antibody does not recognize the CONH~2~ group.

In the current study, we detected kisspeptin precursor in OVA by western, but not by immunofluorescence. This may be due to the specific structures and components of oocytes, as well as the characteristics of the antibody. The presence of the ligand in widely distributed secretory vesicles, which is usually involved in kisspeptin processing and transporting (Hiroko Murakawa, et al., 2016), may be difficult to detect in big oocytes (about 150-200 μm in diameter) by confocal microscopy. For western blotting, the lipids and associated proteins (main components) in mature oocytes are eliminated during sample preparation, consequently increases the concentration of kisspeptin, making it detectable by western blot. Alternatively, it may be also due to the characteristics of the antibody. In fact, many commercially available antibodies are only good for western, not for histochemistry. We explained this inconsistence in revised main text as "the inconsistency with results from the western blotting assay vs. immunofluorescence may be either due to specific structures and components of oocytes, or the characteristics of the antibody".

Murakawa, H., Iwata, K., Takeshita, T., and Ozawa, H. (2016). Immunoelectron microscopic observation of the subcellular localization of kisspeptin, neurokinin B and dynorphin A in KNDy neurons in the arcuate nucleus of the female rat.Neuroscience Letters, 612, 161-166. doi: 10.1016/j.neulet.2015.12.008

> 6\) There are further major omissions of explanations throughout the text, e.g.:
>
> In Figure 4A, the authors completely omit to discuss the PTX treatment shown in the panels. Does PTX stand for Pertussis toxin? The authors need to mention this treatment in the text and explain the reason to use PTX.

We apologize for this omission. PTX does stand for Pertussis toxin. We used PTX to exclude the involvement of Gi protein in the AjKiss receptors-mediated signaling. We have amended the text accordingly.

> Explain AjKiss1b-10 in the text. Same for in the Figure 4 legend. Please consider that not all readers are familiar with the literature on Kisspeptins or neuropeptides in general. Also, please, pick one naming system and keep using it; i.e., use either Kp or Kiss or Kisspeptin. Same for receptor naming.

As the reviewers suggested, we have listed the detailed information of AjKiss1b-10 and pep234, as well as other peptides mentioned in this article in Key Resources Table, and also unified usage of Kiss and KissR in the revised version of manuscript.

> Subsection "In silico identification of Kps and Kp receptors", first paragraph: is that the same sequence identified by Swansa-ard et al/Chen et al? If yes state that and those lines can be omitted as it also repeats the Introduction.

The lines have been removed in the revised manuscript as the reviewers suggested.

> Figure 5A1. What time (5 min, 10 min, etc. stimulation) the concentration-dependence blots refer to?

We challenged the cells with different concentrations of ligand for 5 min in the concentration-dependence blots, and we have provided this info in the revised manuscript.

> Figure 5A2 (bar graph). Why isn\'t the strongest signal for AjKissR2 set at 100% as in the case of AjKissR1?

As shown in the now revised Figure 6A1, AjKissR1 exhibited higher efficacy and potency than that of AjKissR2, therefore, when we deal with the data of Figure 5A2 (bar graph), the ratio of p-ERK1/2/total ERK1/2 was normalized to peak value detected in corresponding experiments and the strongest signal of AjKissR1 was set as 100%, so that we can easily compare the ERK1/2 activity mediated by AjKissR1 and AjKissR2.

> Figure 6D. Upper left panel. What treatment is this?

As the reviewers pointed out, we have clarified the treatment for upper left panel of the now revised Figure 7D.

> Figure 6F. Explain what tissue index measures.

In the now revised Figure 7F, "tissue index" refers to the ratio of tissue weight/body weight. The detailed information of calculation, as well as the statistics, is listed in metadata.

> Figure 7: Why does this figure indicate presence of only one kisspeptin receptor in sea cucumber if the presented study finds two?

We apologize for the omission. We have redrawn this figure (Figure 8 in the revised version) to provide more accurate information about the current discovery about the kiss system.

> Substantial editing work is needed to allow readers to more easily follow the data. Generally, the figure legends and the Materials and methods section need the most attention.

As the reviewers suggested, we have carefully gone through the whole manuscript and corrected typos and grammatical errors, especially in the figure legends and the Materials and methods section.

> Figures. Make larger panels, please. Some of the labels on the panels were very difficult, if not impossible, to decipher (for instance, 1C, 1E, 5A, 5B, 5C). Check for typos in the Figure labels (for instance, Figure 2B: 1 μM instead of 1 um).

We have enlarged figure panels together with the label, and also corrected typos in the figure label as the reviewers suggested.

> Materials and methods.
>
> Animals should not be listed as \"Materials\". Transient and stable HEK293 cells expressing AjKissRs were generated, but it is not explained why. A protocol for DiI staining (Figure 2A) is not provided.

As the reviewers suggested, we have added the animals-related content to the "Animal collection and treatment" subtitle following the Key Resources Table in Materials and methods section. Establishment of stable expression cell lines can reduce the influence caused by different transfection efficiency. We have also added description of the DiI staining protocol in the Materials and methods section.

> Receptor internalization (shown in Figure 2C) mentions 30 min, but the legend says 60 min. In the same experiments, it is said that cells were incubated with DAPI for several minutes before translocation. DAPI is not a vital stain; did the authors mean Hoechst? Species name: sometimes *A. japonicas*, at others it is *A. japonicus*.

We have to apologize for our carelessness in preparing our manuscript, and we have provided more detailed information about DiI and DAPI treatment in the Materials and methods section of revised manuscript as the reviewers suggested. By the way, just like Hoechst, DAPI is a popular nuclear and chromosome counterstain, and emits blue fluorescence upon binding to AT regions of DNA (Biotech Histochem.1995;70(5):220-33; Cell2006;125(4):679-90). Additionally, we have also unified the usage of "*A. japonicus*" in the whole revised manuscript. We thank the reviewers for these suggestions.

> Subsection "Data statistics": please refer to the exact experiments for which these statistic methods were used.

As the reviewers suggested, we have added the description on data statistics in the legend of the exact experiments for which these statistic methods were used in the revised manuscript, and thank the reviewers for this suggestion.

> 7\) In general, a conclusive sentence about what each of the experiments shows would greatly help the reader, e.g.- Subsection "Physiological functions of the Kp signaling system in *A. japonicus*", third paragraph: a one line conclusion on kiss signalling functions inferred from the effects of persistent kiss1a/1b treatment would be nice.
>
> Do you think it maintains metabolic balance, or does it lead to the mobilisation of lipids from the fat tissues?

As the reviewers constructively suggested, we have added a conclusive sentence "These data suggest that the *A. japonicus* kisspeptin system plays a role in the control of metabolic balance". As for the exact physiological role of the *A. japonicus* kisspeptin system, e.g. mobilisation of lipids from the fat tissues, it is in our future research plan.

> 8\) The data on the potential function in seasonal control of reproduction are interesting, but as presented at present rather confusing, as they seem to be partly somewhat contradictory.
>
> The statements in the second and last paragraphs of the subsection "Physiological functions of the Kp signaling system in *A. japonicus*, would suggest an important role in regulating the maturation of the germline, especially oocytes before/during the peak season of reproduction. How does this fit with the findings described in the third paragraph of the aforementioned subsection, describing what rather seems to be a role in aestivation?

As the reviewers pointed out, in the subsection "Physiological functions of the kisspeptin signaling system in *A. japonicus*", we intended to describe the role of kiss system in the metabolic control of sea cucumber. In sea cucumber, aestivation is directly related to metabolism and reproduction, and PK expression and activity is an important physiological index for metabolism and reproduction. Although we don't have strong evidence to support the functional role of kisspeptin in aestivation of sea cucumber, our data indicate the important role of kisspeptin in the regulation of reproductive development and the metabolic balance of sea cucumber. These two different physiological processes arise simultaneously in the seasonal reproduction of *Apostichopus japonicus* (Ru et al., 2017 and 2018). Moreover, the latest data from Ru's research (data unpublished, paper under revision) indicate that reproduction causes a significant increase in oxygen consumption in *A. japonicus*, and sea cucumber can accommodate the high oxygen demand by accelerating respiratory rate (this is consistent with the elevation of *PK* RNA expression level of respiratory tree under kisspeptin administration). Meanwhile, Ru's research also found the decreased appetite during reproductive development which should be related with the degeneration of intestine. Collectively, we believe that kisspeptin administration induces both metabolic regulation (expressional elevation of *PK* and degeneration of intestine) and reproductive control (expression of kisspeptin and its receptor in germline).

Ru, X., Zhang, L., Liu, S., and Yang, H. (2017). Reproduction affects locomotor behaviour and muscle physiology in the sea cucumber, *Apostichopus japonicus*. Animal Behaviour, 133, 223--228. doi:10.1016/j.anbehav.2017.09.024

Ru, X., Zhang, L., Liu, S., Sun, J., and Yang, H. (2018). Energy budget adjustment of sea cucumber *Apostichopus japonicus* during breeding period. Aquaculture Research, 49(4), 1657--1663. doi:10.1111/are.13621

Unpublished paper: Plasticity of respiratory function accommodates high oxygen demand in breeding sea cucumbers

> 9\) Final conclusion: The statement that it \"originally evolved in this hypothalamic neuropeptide system\" implies that it evolved along with the hypothalamus in higher vertebrates. The work presented by the authors does not support this claim, but rather implies that the molecular system itself (including the use of Gaq-PKC cascade in Kp signalling) is likely an ancestral state and is now used by the vertebrate hypothalamus. To draw evolutionary conclusion about an ancestral hypothalamic system the study would have to include other markers for vertebrate hypothalamic cell types (e.g. nkx2.1) and compare how kisspeptin localizes relative to the expression of these other markers. Furthermore, some places of kisspeptin detection- OVA, Tes, ANP- don\'t make sense with an \"ancestral neurosecretory system\" in the sense of an ancestral tissue unit. The authors should revise their text, heading and conclusions according to this, rather pointing out the molecular conservation, as well as the possibly interesting conserved link in the regulation of seasonal reproduction.

As the reviewers suggested, we have removed "originally evolved in this hypothalamic neuropeptide system", and added one sentence "G~αq~-coupled signaling is highly conserved in the kisspeptin signaling systems from *A. japonicus* to mammals". We have already revised corresponding text, heading and conclusions according to reviewers' suggestion. We thank the reviewers for this constructive suggestion.

> \[Editors\' note: further revisions were suggested prior to acceptance, as described below.\]
>
> The manuscript has been significantly improved by the additional experiments and clarifications, but there are some remaining issues that need to be addressed before acceptance, as outlined below. All aspects can be addressed by text or figure amendments. There are no further experiments needed.
>
> 1\) At present there is no strong evolutionary evidence for this system to homologous to the hypothalamus. We would thus ask to remove the word \"hypothalamic\" from the title.
>
> Title suggestion:
>
> \"Existence and functions of a kisspeptin neuropeptide signaling system in a non-chordate deuterostome species\"

We replaced the word "hypothalamic" with "a" in the title. We thank the reviewers for this suggestion.

> 2\) \"Through the evaluation of Ca^2+^ mobilization and other intracellular signals, we found that *A. japonicus* kisspeptins dramatically activated two receptors (AjKissR1 and AjKissR2), via a GPCR-mediated Gαq/PLC/PKC/MAPK signaling pathway, that have functions corresponding to those of the vertebrate kisspeptin system\". There is something wrong with this statement. Again, the GPCR-mediated Gαq/PLC/PKC/MAPK signaling pathway was shown in a mammalian cell line surrogate. So, while ex vivo experiments show ERK (i.e., MAPK cascade) involvement (Figure 7C and D) the authors have not shown that in/ex vivo Aj kisspeptin signaling involves PLC and PKC.
>
> We would thus suggest to rephrase this in the following way (or something similar):
>
> \"Through the evaluation of Ca^2+^ mobilization and other intracellular signals, we found that *A. japonicus* kisspeptins activate two receptors (AjKissR1 and AjKissR2) via a GPCR-mediated Gαq/PLC/PKC/MAPK signaling pathway in a mammalian cell line. Albeit likely it remains to be shown if the same signaling cascade also occurs in vivo in its seemingly conserved function in reproductive control.\"

As the reviewers suggested, we have rephrased these sentences in the revised version. We thank the reviewers for this suggestion.

> 3\) Subsection "In silico identification of kisspeptins and kisspeptin receptors". Change \"ANP, containing nerve ring\" to \"ANP, containing the nerve ring\".

We have added "the" to this sentence. We thank the reviewers.

> 4\) Subsection "In silico identification of kisspeptins and kisspeptin receptors". Change \"frames\" to \"frame\".

We have corrected it in revision and thank the reviewers.

> 5\) Figure 3B legend. Change \"3-B\" to \"3B\".

The "-" has been deleted. We thank the reviewers.

> 6\) Figure 4 legend. It recalls the Figure 4 source data. Here, there is a mislabelling, as the linked excel files has individual worksheet labelled as 3A, 3B etc. -- they should be 4A, 4B etc, instead. The authors should check all their supplementary files for correct labelling.

We have corrected these labels and checked all the labels in our supplementary files. Besides, an error of data in the source data sheet of "Figure 7H FEB" (cells C27-C29) was found and the source data file (Figure 7E+F+H metadata), as well as the heatmap in Figure 7, was corrected accordingly in revision. We apologize for the carelessness in writing and data organization. We thank the reviewers.

> 7\) \"inhibitory effect of pep234 was preapproved in vitro\", should be changed to \"inhibitory effect of pep234 was validated in vitro\".

The sentence has been optimized. We thank the reviewers.

> 8\) \"Further detection of the pERK signal in AjKiss1b-10 treated oocytes by confocal microscopy demonstrated the physiological activation of this pathway by AjKiss1b-10 and pep234 on *A. japonicus* cells\" should be changed to \"Further, detection by confocal microscopy of the p-ERK signal in treated oocytes demonstrated activation of this pathway by AjKiss1b-10 and its inhibition by pep234 in *A. japonicus* cells\".

We have rewritten the sentence following the reviewers' comments. We thank the reviewers.

> 9\) \"Samples were collected and fixed after 2 h of ligand administration with or without a 4 h pre-treatment of pep234, in optimized L15 medium at 18 °C\". Should be changed to \"Samples were collected and fixed after 2 h of ligand administration with or without a 4 h pre-treatment with pep234, in optimized L15 medium at 18 °C\". Also, is this the same treatment for the samples shown in Figure 7C? If so, it should be clearly stated in the legend for panel 7C.

The legend has been revised following reviewers' comments. We thank the reviewers.

> 10\) After \"different potency\", we suggest the addition of \", when expressed in a mammalian cell line\".

The sentence has been revised following reviewers' comments. We thank the reviewers.

> 11\) Change statement from \"therefore proving the functionality of this neuropeptide system in non-chordate species\" to \"therefore proving that this peptide system is present and active in non-chordate deuterostome species\".

The sentence has been revised following reviewers' comments. We thank the reviewers.

> 12\) \"an amphioxus kisspeptin receptor was shown to trigger significant PKC and not PKA signaling, when stimulated by two kisspeptin-type peptides (Wang et al., 2017).\" Please, make clear that these experiments too were done using heterologous expression in HEK293 cells.

An additional description "using heterologous expression in cultured HEK293 cells" was added to this sentence in text. We thank the reviewers.

> 13\) It is stated that \"AjKissR1 and AjKissR2 induced ERK1/2 activation via a Gαq/PLC/PKC cascade. Our results showed that no significant accumulation of cAMP was detected in response to agonist treatment\". We could not find any experiment in the manuscript measuring cAMP accumulation. The authors should amend the text accordingly.

This statement has been removed and an additional sentence "we failed to collect distinct evidence to prove the involvement of G~αs~-dependent signaling in *A. japonicus* Kisspeptin system." was added following "Although the G~αs~ protein has been shown to be implicated in the teleost kisspeptin receptors-mediated signaling pathway,". These sentences were amended accordingly. We thank the reviewers.

> 14\) All images of the Western blots are over-contrasted. This needs to be corrected and size markers also added to Figure 7 (not just the figure supplement).

We have checked and revised all images of the Western blots as the reviewers suggested. Besides, we found that the WB images in Figure 7---figure supplement 3B, for the inhibitory activity of pep234 in AjKissR2 mediated ERK signaling, was upside down in the previous version. We have also corrected the data in revision. We thank the reviewers for pointing out this issue.

> 15\) Carefully check for missing and correct scalebars. Figure 3: According to the scale bar these HEK cells are huge. Sure the scale bar is correct? Figure 6D, E: scale bars are entirely missing.

We apologize for the incorrect and missing scalebars. We have checked all the scalebars referring to primary data. The "20 μm" in Figure 3 was marked by mistake, and it has been revised as "10 μm". The scalebars in Figure 6 have been added. Meanwhile, the scale bars in Figure 6---figure supplement 1 were adjusted too. We thank the reviewers for pointing out this issue.

> 16\) There is still some remaining concern about the specificity of the antibody against AjKiss1b. We find it pretty unusual that an antibody that was generated against an amidated peptide would now only rather recognize the precursor (or if we played devil\'s advocate- a much larger band in the Western that could have nothing to do with the actual peptide). We would thus ask the authors to insert a cautionary sentence in the text.

Thank the reviewers for this comment. We have added a cautionary sentence, "However, the failure to detect mature peptide fragment using anti-AjKiss1b-10 indicates that the further development of antibodies with highsensitivityandspecificity might be required to clarify the mature kisspeptin location in *A. japonicus* tissues. " following suggestion.

[^1]: These authors contributed equally to this work.
