Comparative and Evolutionary Aspects of Gonadotropin-Inhibitory Hormone and FMRFamide-Like Peptide Systems

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that was found in the brain of Japanese quail when investigating the existence of RFamide peptides in birds. GnIH was named because it decreased gonadotropin release from cultured anterior pituitary, which was located in the hypothalamo-hypophysial system. GnIH and GnIH precursor gene related peptides have a characteristic C-terminal LPXRFamide (X = L or Q) motif that is conserved in jawed vertebrates. Orthologous peptides to GnIH are also named RFamide related peptide or LPXRFamide peptide from their structure. A G-protein coupled receptor GPR147 is the primary receptor for GnIH. Similarity-based clustering of neuropeptide precursors in metazoan species indicates that GnIH precursor of vertebrates is evolutionarily related to FMRFamide precursor of mollusk and nematode. FMRFamide peptide is the first RFamide peptide that was identified from the ganglia of the venus clam. In order to infer the evolutionary history of the GnIH-GnIH receptor system we investigate the structural similarities between GnIH and its receptor and well-studied nematode Caenorhabditis elegans (C. elegans) FMRFamide-like peptides (FLPs) and their receptors. We also compare the functions of FLPs of nematode with GnIH of chordates. A multiple sequence alignment and phylogenetic analyses of GnIH, neuropeptide FF (NPFF), a paralogous peptide of GnIH, and FLP precursors have shown that GnIH and NPFF precursors belong to different clades and some FLP precursors have structural similarities to either precursor. The peptide coding regions of FLP precursors in the same clade align well with those of GnIH or NPFF precursors. Alignment of GnIH (LPXRFa) peptides of chordates and FLPs of C. elegans grouped the peptides into five groups according to the last C-terminal amino acid sequences, which were MRFa, LRFa, VRFa, IRFa, and PQRFa. Phylogenetic analysis of receptors suggested that GPR147 has evolutionary relationships with FLP receptors, which regulate reproduction, aggression, locomotion, and feeding. GnIH and some FLPs mediate the effect of stress on reproduction and behavior, which may also be a conserved property of these peptide systems. Future studies are needed to investigate the mechanism of how neuropeptide precursor genes are mutated to evolve new neuropeptides and their inheritance.

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that was found in the brain of Japanese quail when investigating the existence of RFamide peptides in birds. GnIH was named because it decreased gonadotropin release from cultured anterior pituitary, which was located in the hypothalamo-hypophysial system. GnIH and GnIH precursor gene related peptides have a characteristic C-terminal LPXRFamide (X = L or Q) motif that is conserved in jawed vertebrates. Orthologous peptides to GnIH are also named RFamide related peptide or LPXRFamide peptide from their structure. A G-protein coupled receptor GPR147 is the primary receptor for GnIH. Similarity-based clustering of neuropeptide precursors in metazoan species indicates that GnIH precursor of vertebrates is evolutionarily related to FMRFamide precursor of mollusk and nematode. FMRFamide peptide is the first RFamide peptide that was identified from the ganglia of the venus clam. In order to infer the evolutionary history of the GnIH-GnIH receptor system we investigate the structural similarities between GnIH and its receptor and well-studied nematode Caenorhabditis elegans (C. elegans) FMRFamide-like peptides (FLPs) and their receptors. We also compare the functions of FLPs of nematode with GnIH of chordates. A multiple sequence alignment and phylogenetic analyses of GnIH, neuropeptide FF (NPFF), a paralogous peptide of GnIH, and FLP precursors have shown that GnIH and NPFF precursors belong to different clades and some FLP precursors have structural similarities to either precursor. The peptide coding regions of FLP precursors in the same clade align well with those of GnIH or NPFF precursors. Alignment of GnIH (LPXRFa) peptides of chordates and FLPs of C. elegans grouped the peptides into five groups according to the last C-terminal amino acid sequences, which were MRFa, LRFa, VRFa, IRFa, and PQRFa. Phylogenetic analysis of receptors suggested that GPR147 has evolutionary relationships with FLP receptors, which regulate reproduction, aggression, locomotion, and feeding. GnIH and some FLPs mediate the effect of stress on reproduction and behavior, which may also be a conserved property of these peptide systems. Future studies are needed to investigate the mechanism of how neuropeptide precursor genes are mutated to evolve new neuropeptides and their inheritance.

INTRODUCTION
A hypothalamic neuropeptide gonadotropin-inhibitory hormone (GnIH) was discovered in the Japanese quail (Coturnix japonica) brain, while studying the existence of Arg-Phe-NH 2 (RFamide) peptides in birds, which have a characteristic C-terminal RFamide sequence (Tsutsui et al., 2000). Phe-Met-Arg-Phe-NH 2 (FMRFamide) is the first RFamide peptide that was identified in the venus clam Macrocallista nimbosa ganglia, which has a cardioexcitatory function (Price and Greenberg, 1977). Since then, multiple RFamide peptides acting as hormones, neuromodulators and neurotransmitters have been found in cnidarians, nematodes, annelids, mollusks, and arthropods. Multiple immunohistochemical studies using antibodies against RFamide peptides of invertebrates suggested the presence of RFamide peptides in the central nervous system of vertebrates. Tsutsui et al. (2000) have successfully isolated a peptide from 500 Japanese quail brains using high-performance liquid chromatography combined with a competitive enzyme-linked immunosorbent assay with an antibody against Arg-Phe-NH 2 . The C-terminal structure of the isolated peptide SIKPSAYLPLRFamide ( Table 1) was found to be identical to the chicken LPLRFamide that was reported as the first RFamide peptide isolated in vertebrates (Dockray et al., 1983). However, the previously reported chicken LPLRFamide peptide can be the fragment of the chicken GnIH peptide that was identified to have a sequence of SIRPSAYLPLRFamide in a recent study (McConn et al., 2014). GnIH was named "gonadotropininhibitory hormone" because it decreased gonadotropin release from cultured quail anterior pituitary gland and located in the hypothalamo-hypophysial system (Tsutsui et al., 2000; for reviews see, Tsutsui et al., 2015Tsutsui et al., , 2017Ubuka et al., 2016).

STRUCTURE OF GNIH PEPTIDES AND THEIR POSITIONS IN THEIR PRECURSOR PROTEINS
In the following year of the discovery of quail GnIH, the precursor cDNA of quail GnIH was cloned and sequenced (Satake et al., 2001). GnIH precursor is composed of 173 amino acid residues encompassing GnIH as well as two GnIH-related peptides (GnIH-RP-1 and GnIH-RP-2;  Table 1). GnIH and GnIH-related peptide sequences are flanked by an amidation signal glycine at the C-terminal as well as basic amino acids (arginine or lysine) as endoproteolytic sites on each end (Supplementary Figure 1). The translated and processed GnIH and GnIH-RPs all possess a C-terminal LPXRFamide (X = L or Q) sequence (Figure 1,  Supplementary Figure 1, Table 1). Mass spectrometry has also identified the mature peptide structure of GnIH-RP-2 in addition to GnIH (Satake et al., 2001). Within the class of birds, mature GnIH peptides were also isolated in European starlings , zebra finch , and chicken (McConn et al., 2014;Ubuka et al., 2016). A cDNA encoding LPXRFamide peptides in the brain of red-eared slider was recently cloned and immunoaffinity purification and mass spectrometry identified three mature LPXRFamide peptides that were encoded in the precursor protein (Ukena et al., 2016).

GNIH RECEPTOR AND CELL SIGNALING
GnIH (RFRPs) suppresses cAMP production in cells transfected with chicken and rat GPR147 suggesting that GPR147 couples to G αi protein which inhibits adenylate cyclase (AC) (Hinuma et al., 2000;Shimizu and Bédécarrats, 2010). The GnIH cell signaling pathway has been precisely investigated in LβT2 cells, a mouse gonadotrope cell line . Mouse GnIHs (RFRPs) suppressed gonadotropin-releasing hormone (GnRH)-induced cAMP signaling, extracellular signalregulated kinase (ERK) phosphorylation as well as gonadotropin subunit gene transcription by inhibiting the protein kinase A (PKA) pathway Ubuka et al., 2013). Because GnIH neurons extend their axons to GnRH neurons and GnRH neurons express GPR147 in birds and mammals , GnIH cell signaling pathway was further investigated in GT1-7, a mouse GnRH neuronal cell line . It was found that GnIH suppresses the effect of vasoactive intestinal polypeptide on AC activity, p38 and ERK phosphorylation, and c-Fos mRNA expression in GT1-7 cells . These results suggest that GnIH specifically inhibits the AC/cAMP/PKA pathway in gonadotropes and GnRH neurons at least in birds and mammals .
GnIH (RFRP-3) rapidly and repeatedly inhibits the firing of GnRH neurons as well, which was shown in the adult mice . It was further shown that GnIH (RFRP-3) produces a non-desensitizing hyperpolarization in vesicular glutamate transporter 2 (vGluT2)-GnRH neurons by a direct postsynaptic Ba 2+ -sensitive K + current mechanism ).
GnIH neuronal cell bodies are clustered in the medial hypothalamic area, but in different brain regions or nuclei in mammals. In hamsters and mice, a cluster of GnIH neuronal cell bodies exists in the dorsomedial hypothalamic area (DMH) Ubuka et al., 2012a), whereas in rats it is in the periventricular nucleus (PerVN), and the portion between the dorsomedial (DMN) and the ventromedial (VMN) nuclei of the hypothalamus (Hinuma et al., 2000;Legagneux et al., 2009). In the macaque brain, a cluster of GnIH neuronal cell bodies principally exists in the intermediate periventricular nucleus (IPe) of the hypothalamus (Ubuka et al., 2009b), whereas in sheep it exists in the DMN and PVN . GnIH-ir neuronal fibers are also widely located in the diencephalic, mesencephalic and limbic brain regions in mammals (Yano et al., 2003;Johnson et al., 2007;Ubuka et al., 2009bUbuka et al., , 2012a. Studies in macaque, sheep and mice showed that GnIH-ir fibers are in close proximity to GnRH-1, dopamine, proopiomelanocortin (POMC), GnRH-2, neuropeptide Y (NPY), orexin, melanin-concentrating hormone (MCH), corticotrophinreleasing hormone (CRH), oxytocin, and kisspeptin neurons Ubuka et al., 2009b;Poling et al., 2013). Five to ten percentage of kisspeptin neurons in the anteroventral periventricular (AVPV) region express GPR147 or GPR74, whereas approximately 25% express GPR147 or GPR74 in kisspeptin neurons in the arcuate nucleus in mice .
GnIH neuronal cell bodies are located in the nucleus accumbens, PVN, and upper medulla, and fibers contact the lateral processes of serotonin-ir neurons in the paraventricular organ (PVO) in the Japanese grass lizard (Kawano et al., 2006). On the other hand, GnIH neuronal cell bodies are restricted to the periventricular hypothalamic nucleus, and the fibers are densely distributed in the median eminence of red-eared slider turtle (Ukena et al., 2016).
GnIH neuronal cell bodies constitute two subpopulations in the telencephalon and diencephalon and the highest number of cell bodies are located in the POA and suprachiasmatic areas of the anuran amphibian Rana esculenta . GnIH neuronal cell bodies also exist in the medial septum, anterior commissure, dorsal hypothalamus, PerVN of the hypothalamus, and posterior tuberculum. GnIH neuronal fibers are only occasionally present in the median eminence. GnIH neuronal fibers exist in close proximity to GnRH cell bodies . GnIH (LPXRFa) neuronal cell bodies are located in the nucleus posterioris periventricularis and the nervus terminalis, and the fibers extend to nucleus lateralis tuberis pars posterioris and pituitary in goldfish and sockeye salmon (Sawada et al., 2002b;Amano et al., 2006). Sea bass GnIH (sbLPXRFa) neuronal cell bodies exist in the olfactory bulbs-terminal nerve, ventral telencephalon, caudal POA, dorsal mesencephalic tegmentum and rostral rhombencephalon, and fibers are widely distributed including the pituitary. GnIH (sbLPXRFa) neuronal fibers exist close to luteinizing hormone (LH), follicle-stimulating hormone (FSH), and growth hormone (GH) cells .
By controlling gonadotropin secretion, GnIH regulates reproductive development and maintenance in birds (Ubuka

Regulation of GH Secretion
GnIH (RFRP-3) increases GH-releasing hormone mRNA expression in the hypothalamus and GH release in rats (Johnson et al., 2007;Johnson and Fraley, 2008). In bullfrog, LPXRFamide peptide (fGRP) also stimulates GH release (Koda et al., 2002). On the other hand, GnIH (LPXRFa peptide) has both stimulatory and inhibitory effects on GH release and/or expression in teleost fishes Moussavi et al., 2014). Goldfish GnIH (gfLPXRFa) peptides stimulate GH release from cultured sockeye salmon pituitary cells . On the other hand, injection of gfLPXRFa to goldfish reduces basal serum GH levels but increases pituitary GH mRNA levels . Injection of gfLPXRFa reduces serum GH and pituitary GH mRNA levels stimulated by goldfish GnRH (sGnRH and cGnRH-II) . However, administration of gfLPXRFa to goldfish pituitary cells for 24-h generally increases basal GH release and attenuates sGnRH-induced changes in GH mRNA depending on the reproductive stage . These results indicate that the effect of GnIH (LPXRFa peptide) on GH release and/or expression depends on reproductive condition in teleost fishes.

Regulation of Cardiac Contraction
Although expression levels of GnIH and GPR147 mRNAs are under detectable levels in the heart of rats, it was reported that human RFRP-1 and rat RFRP-1 rapidly and reversibly decrease the shortening and relaxation of cardiac myocytes in rats and rabbits (Nichols et al., 2010(Nichols et al., , 2012. Human RFRP-1 decreases the heart rate, stroke volume, ejection fraction, as well as cardiac output in mice (Nichols et al., 2010). It was suggested that human RFRP-1 impairs myocyte shortening by enhancing myofilament protein phosphorylation by protein kinase C (Nichols et al., 2012).

Regulation of Stress Responses
Acute and chronic immobilization stress both up-regulates GnIH expression in the DMH of rats associated with inhibition of downstream HPG activity (Kirby et al., 2009). Endotoxin administration increases GnIH and GPR147 mRNA expression in rats . The effect of short-term fasting and high-fat diet on gonadotropin suppression was less effective in GPR147-deficient male mice, suggesting the involvement of GnIH-GPR147 pathway in the suppression of gonadotropin secretion by metabolic stress . Capturehandling stress and high ambient temperature increase GnIH expression in house sparrows (Calisi et al., 2008) and chicks , respectively. Female presence also increases GnIH mRNA expression and GnIH release by rapid increase in norepinephrine in the PVN in male quail . It was demonstrated that corticosterone increases GnIH mRNA expression via glucocorticoid receptor expressed in GnIH neurons in birds and mammals (Ahmed et al., 2014;Son et al., 2014). Neonatal dexamethasone exposure increases GnIH cell numbers in the DMH and GPR147 mRNA in the POA in female mice with delayed vaginal opening, irregular estrous cycles and lower GnRH expression in the POA  Biochemically identified mature peptides are shown in bold (Peymen et al., 2014). Peptide names are from Krajniak (2013). Numbers of peptides are shown if the same peptide is encoded in the precursor. (Soga et al., 2012). GnIH (RFRP) administration induces anxietyrelated behavior in rats (Kaewwongse et al., 2011), suggesting that GnIH also mediates behavioral stress responses (Ubuka et al., 2018).

Regulation of Reproductive and Aggressive Behaviors
GnIH (RFRP-3) suppresses sexual behavior of male rats (Johnson et al., 2007) and proceptive sexual behavior and motivation of female hamsters (Piekarski et al., 2013). GnIH also inhibits copulation solicitation of female white-crowned sparrows exposed to male song . RNA interference (RNAi) of the GnIH gene (GnIH RNAi) reduces resting time, spontaneous production of complex vocalizations, and enhances spontaneous brief agonistic vocalizations and song production of short duration in male birds when they were exposed to playbacks of novel male songs in white-crowned sparrows, suggesting that GnIH suppresses locomotor activity and aggressiveness (Ubuka et al., 2012b). GnIH directly activates P450arom and increases neuroestrogen synthesis in the brain and suppresses socio-sexual behavior of male birds .

Regulation of Feeding Behavior
GnIH (RFRP-3) increases food intake in male rats (Johnson et al., 2007) and sheep . GnIH mRNA levels are decreased in adult obese mice of both sexes . GnIH also stimulates food intake in chicks (Tachibana et al., 2005(Tachibana et al., , 2008McConn et al., 2014) and adult Pekin drakes see, Tsutsui and Ubuka, 2016) for a review). Studies in chicks suggested that the orexigenic effects of GnIH involves NPY, MCH, POMC neurons and opioid mu-receptor in the brain (Tachibana et al., 2008;McConn et al., 2014). Histological and physiological studies showed that NPY and POMC neurons are also regulated by GnIH in mammals Ubuka et al., 2009b;Fu and van den Pol, 2010;Jacobi et al., 2013). The stimulatory effect of stress on GnIH neurons as well as the effect of GnIH on gonadotropin secretion, GH synthesis and release, and feeding activities suggest that GnIH neurones coordinate reproduction, growth and feeding activities in response to stress.

Gonadal GnIH
GnIH and GPR147 exist in the testis of Syrian hamster  and the ovary of mice  and humans . GnIH (RFRP-3) suppresses spermatogenesis, follicular development and/or steroidogenesis in mice Anjum et al., 2014), pigs , and humans . GnIH and GPR147 also exist in the gonads and accessory reproductive organs in passeriform and galliform birds

Evolutionary History of GnIH and Its Receptor
We previously searched for receptors that are structurally similar to GPR147 in the genome of mammals, birds, reptiles, amphibians, fishes, hemichordates, echinoderms, mollusks, insects, and cnidarians, to infer the evolutionary history of the GnIH system . Neighbor joining (NJ) and maximum likelihood (ML) analyses of the amino acid sequences of the receptors grouped the receptors of vertebrates into GPR147 and GPR74. The receptors of insects were grouped into the receptor for SIFamide peptides that have a C-terminal YRKPPFNGSIFamide motif . Human, quail, and zebrafish GPR147 was most structurally similar to SIFamide receptor within the C-terminal Famide peptide (SIFamide, FMRFamide, neuropeptide F, short neuropeptide F, drosulfakinin, myosuppressin) receptor families of fruit fly . On the other hand, the amino acid sequences and the peptide coding regions of GnIH precursors were most similar to the FMRFamide precursor of fruit fly . Chromosome synteny analysis of human, quail and zebrafish GnIH precursor genes and fruit fly Famide peptides precursor genes further identified a conserved synteny in vertebrate GnIH and fruit fly FMRFamide peptide precursor genes . These results suggest that GnIH and its receptor pair have evolved from ancestral FMRFamide peptide and its receptor pair FIGURE 3 | A phylogenetic analysis of human, quail, newt, coelacanth, zebrafish, gar, lamprey, amphioxus GnIH, human, quail, turtle, zebrafish, gar, lamprey, amphioxus NPFF, fruit fly FMRFamide and C. elegans FMRFamide-like peptide (FLP) precursors. Human, quail, newt, coelacanth, zebrafish, gar, lamprey, amphioxus GnIH, human, quail, turtle, zebrafish, gar, lamprey, amphioxus NPFF, fruit fly FMRFamide, and C. elegans FLP precursor polypeptides were aligned by CLUSTALW Multiple Sequence Alignment. Multiple alignment parameters were as follows: Gap open penalty 10, Gap extension penalty 0.2, Protein weight matrix GONNET with residue-specific and hydrophilic penalties. Molecular phylogenetic analysis was performed by Maximum Likelihood method using MEGA7 (Kumar et al., 2016). The Maximum Likelihood method was based on the JTT matrix-based model (Jones et al., 1992). The tree with the highest log likelihood is shown. Initial tree for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated by using a JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn with branch lengths measured in the number of substitutions per site. The analysis involved 51 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 32 positions in the final dataset. The phylogeny was tested by 50 Bootstrap replications. Accession numbers are human (Homo sapiens) GnIH precursor (Human GnIH; NP_071433.3), Japanese quail (Coturnix japonica) GnIH precursor (Quail GnIH; XP_015709159.1), Japanese fire belly newt (Cynops pyrrhogaster) GnIH precursor (Newt LPXRFamide peptide; BAJ78290.1), West Indian Ocean coelacanth (Latimeria chalumnae) GnIH precursor (Coelacanth LPXRFamide peptide; XP_005993154.1), zebrafish (Danio rerio) GnIH precursor (Zebrafish LPXRFamide peptide, NP_001076418.1), spotted gar (Lepisosteus oculatus) GnIH precursor (Gar LPXRFamide peptide; XP_015213317.1), sea lamprey (Petromyzon marinus) GnIH precursor (Petromyzon marinus LPXRFamide peptide; BAL52329.1), Japanese amphioxus (Branchiostoma japonicum) GnIH precursor (Branchiostoma japonicum RFamide peptide; BAO77760.1), human NPFF precursor isoform 1 (Human NPFF isoform 1; NP_003708.1), human NPFF precursor isoform 2 (Human NPFF isoform 2; NP_001307225.1), Japanese quail NPFF precursor (Quail NPFF; XP_015705838.1), Western painted turtle (Chrysemys picta bellii) NPFF precursor (Turtle NPFF; XP_005307776.1), zebrafish NPFF precursor (Zebrafish NPFF; BAF34891.1), spotted gar NPFF precursor isoform X2 (Gar NPFF isoform 2; XP_015199730.1), sea lamprey NPFF precursor (Petromyzon marinus PQRFamide peptide; BAE79779. during the diversification and evolution of deuterostomian and protostomian species . Similarity-based clustering of various neuropeptide precursors in metazoan species forms one central cluster (Jékely, 2013). The core of the central cluster contains FMRFamide precursor of mollusk and nematode and GnIH precursor of vertebrates is evolutionarily directly related to FMRFamide precursor of mollusk and nematode (Jékely, 2013). FMRFamide-like peptides (FLPs) are the largest family of neuropeptides identified since the FMRFamide peptide was found in the venus clam Macrocallista nimbosa (Price and Greenberg, 1977;McVeigh et al., 2006). In order to further infer the evolutionary history of the GnIH-GnIH receptor system we investigate the structural similarities between GnIH and its receptor and well-studied nematode Caenorhabditis elegans (C. elegans) FLPs and their receptors and compare the functions of FLPs of nematode with GnIH of chordates.

C. elegans FLP SYSTEM
The sequencing of C. elegans genome (The C. elegans Sequencing Consortium, 1998) revealed 119 neuropeptide precursor genes which is subdivided into three major families, 31 flp gene family, 40 insulin-like peptide (ins) gene family, and 48 other neuropeptide-like protein (nlp) genes (Nelson et al., 1998;Li et al., 1999;Kim and Li, 2004;Frooninckx et al., 2012). Each FLP precursor gene of C. elegans encodes one to eight FLPs (Figure 1, Supplementary Figure 1, Table 2). Flp-29 and flp-30 previously reported as parasite specific genes (McVeigh et al., 2006) are orthologous to flp-28 and flp-2, respectively (McCoy et al., 2014). Flp-31 is specific to plant parasitic nematodes (McCoy et al., 2014). Therefore, C. elegans do not have flp-29, 30, 31 ( Table 2). The mature sequences of 41 FLPs were biochemically identified within total 71 FLPs encoded in the 31 flp genes (McCoy et al., 2014;Peymen et al., 2014; Table 2). FMRFamide peptide is not encoded in C. elegans flp genes unlike FIGURE 4 | A phylogenetic analysis of human, quail, newt, coelacanth, zebrafish, gar, lamprey, and amphioxus GnIH (LPXRFa) peptides and C. elegans FMRFamide-like peptides (FLPs). Predicted or biochemically identified endogenous peptides shown in Table 3 were analyzed. The accession numbers of their precursor proteins are shown in the legend of Figure 1. The phylogenetic tree was constructed by Neighbor Joining method (Saitou and Nei, 1987) with the proportion of different sites statistical substitution model using MEGA 7 (Kumar et al., 2016). that of mollusks. The relatedness of FLPs is still unclear because of the large diversity of the FLP sequences (Peymen et al., 2014). It was suggested that flp-27 encodes C-terminal RXRFamide motif that is characteristic of NPF family of invertebrates (Clynen et al., 2009). FLPs are expressed in the majority of 302 neurons including sensory neurons, interneurons, and motor neurons in adult hermaphrodites and they are involved in neuroendocrine activity, locomotion, reproduction, and feeding (Peymen et al., 2014; Table 3). Predicted C. elegans neuropeptide receptors belong to rhodopsin and secretin GPCR families and 9 receptors that belong to rhodopsin family GPCR are activated by FLPs at EC 50 of 1-100 nM (Frooninckx et al., 2012;Peymen et al., 2014; Table 3).

FLP-25-2 ASYDYIRFa
Biochemically identified mature peptide names are shown in bold (Peymen et al., 2014;Ubuka et al., 2016). Characteristic C-terminal sequences that are identical within each group are also shown in bold.
It was shown that FLPs encoded by flp-18 are potent ligands of NPR-5a and NPR-5b, which are the splice variants of npr-5 (Kubiak et al., 2008;Cohen et al., 2009; Table 3). NPR-4 is also activated by FLP-18 peptides (Cohen et al., 2009; Table 3). Flp-18 loss of function or npr-4 and npr-5 deletion mutants display dauer formation, foraging defects, accumulation of excess intestinal fats and reduce aerobic metabolism (Cohen et al., 2009). It is hypothesized that detection of nutrition by sensory neurons (AWC, AFD, ASE) induces FLP-18 peptides release from AIY interneurons. FLP-18 peptides induces fat accumulation by acting on NPR-4 in intestine and NPR-5 in ciliated sensory neurons. NPR-4 in RIV and AVA neurons modulates responses to odor and foraging behavior. FLP-18 peptides also regulate dauer formation by acting on NPR-5 in ASJ neurons (Cohen et al., 2009).
GnIH stimulates feeding behavior in rats (Johnson et al., 2007), sheep , chicks (Tachibana et al., 2005(Tachibana et al., , 2008McConn et al., 2014), and Pekin drakes  and GnIH mRNA expression is reduced in adult obese mice ; see Tsutsui and Ubuka, 2016 for a review). Loss of function of FLP-18 or its receptors NPR-4, NPR-5 induces foraging defects, accumulation of excess intestinal fats and reduction in aerobic metabolism (Kubiak et al., 2008;Cohen et al., 2009; Table 3). Regulation of feeding behavior and metabolism may also be a conserved property of GnIH and FLPs, although the precise mechanism is not understood.

CONCLUSION
In order to infer the evolutionary history of the GnIH-GnIH receptor system, we compared the structures and functions of GnIH and its receptor of chordates with C. elegans FLPs and their receptors. One or two C-terminal LPLRFamide peptides and one to three C-terminal LPQRFamide peptides were encoded in the LPXRFamide (X = L or Q) precursor polypeptide genes of jawed vertebrates (human, quail, newt, coelacanth, zebrafish, gar). Orthologous LPXRFamide precursor polypeptide genes of lamprey and amphioxus encoded only two or three C-terminal PQRFamide peptides. Each FLP precursor gene encodes one to eight FLPs that have generally the same C-terminal sequences especially the last three amino acids. A multiple sequence alignment and phylogenetic analyses of GnIH, NPFF and FLP precursors (Figures 3, 6, Supplementary Figure 2) have shown that GnIH and NPFF precursors belong to different clades and there are FLPs that have structural similarities to either precursor. 3,8,17,18,24,27,and 28 precursors form a clade with GnIH precursors, while FLP-14, 19, and 21 precursors form a different clade with NPFF precursors (Figures 3, 6). Although the peptide coding regions of FLP precursors in the same clade align well with those of GnIH or NPFF precursors, the sequence similarities of the peptides within the aligned precursors were weak (Figure 6). On the other hand, alignment of GnIH (LPXRFa) peptides of chordates and FLPs of C. elegans grouped the peptides into five groups according to the last C-terminal amino acid sequences, which were MRFa, LRFa, VRFa, IRFa, and PQRFa. C-terminal LPLRFamide peptides of jawed vertebrates were all in the LRFa group with other FLPs. On the other hand, C-terminal LPQRFamide peptides of jawed vertebrates and C-terminal PQRFamide peptides of lamprey and amphioxus were grouped in the PQRFa group excluding FLPs.
C-terminal LPLRFamide peptides may be the original form of LPXRFamide (X = L or Q) peptides as many FLPs have the C-terminal LRFa sequence.
Phylogenetic analysis of GnIH receptors and FLP receptors suggested that GPR147 and GPR74 have a strong evolutionary relationship with NPR-22, followed by NPR-11, NPR-1, NPR-5, NPR-4, and NPR-10. It is interesting that these receptors regulate reproduction, locomotion and feeding as GnIH and GPR147. It is also important that NPR-11 and NPR-3 bind FLP-21 and FLP-15-1,-2, respectively, peptides which all have a Cterminal PLRFamide sequence. GnIH and some FLPs mediate the effect of stress on reproduction and behavior, which may also be a conserved property of these peptide systems. Future studies are needed to investigate how neuropeptide precursor genes are mutated to evolve new neuropeptides and their inheritance.

AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.