Identiﬁcation and Characterization of Hdh-FMRF2 Gene in Paciﬁc Abalone and Its Possible Role in Reproduction and Larva Development

: FMRFamide-related peptides are neuropeptides involved in a wide range of biological processes, including reproduction and larval development. To characterize the involvement of FMRFamide in the reproduction and larval development of Paciﬁc abalone Haliotis discus hannai , an FMRFamide cDNA ( Hdh-FMRF2 ) was cloned from the cerebral ganglion (CG). Fluorescence in situ hybridization and qRT-PCR were performed for functional characterization. The Hdh-FMRF2 cDNA encoded 204 deduced amino acids that contained a putative signal peptide and four FaRP domains. The major population of Hdh-FMRF2 neuronal cell bodies was localized in the cortex of CG. Hdh-FMRF2 mRNA expression was signiﬁcantly upregulated in CG during the mature stage of gonadal development and effective accumulative temperature (EAT) exposed abalone in both sexes. In the induced spawning event, Hdh-FMRF2 expression was signiﬁcantly upregulated during spawning in males. However, no upregulation was observed in females, suggesting Hdh-FMRF2 might inhibit gamete release in female abalone. These results revealed Hdh-FMRF2 as a reproduction related peptide. Furthermore, mRNA expression in larval development suggested that this peptide was also involved in larval development during development of Paciﬁc abalone. Collectively, this study provides evidence of possible involvement of an FMRFamide neuropeptide in the reproduction and larval development of Paciﬁc abalone.

FaRPs are the most comprehensively studied neuropeptides in invertebrates [10][11][12] with tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH2; FMRFa) being one of the most representative members of this peptide family. FaRPs share a high sequence similarity with FMRFamide and are evolutionarily related to FMRFamide. The first FaRP, an FMRFamide was identified in the sunray venus clam (Macrocallista nimbosa), a bivalve mollusk species [5]. Since then, the sequence, structure, localization and physiological effects of FaRPs have been widely studied in cephalopods, gastropods, and other species [2,7,[13][14][15][16][17]. More than 40 types of FaRPs have thus far been identified in the 5 major mollusk classes [14,18]. Among gastropod mollusks, the FMRFamide gene has been studied extensively in pond snail [11,17] and sea hare [14,16]. In abalone species, two transcripts of FMRFamide have tissues were then sectioned into 6 µM thick slices in transverse orientation using a cryostat microtome (Leica CM3050; Wetzlar, Germany). The sections were fixed on SuperFrost™ Plus slides (Fisher Scientific, Pittsburgh, PA, USA) and stored at −20 • C until required.

Tissue Collection for mRNA Expression Analysis in Different Experimental Conditions
Tissue samples were collected at different experimental conditions. Prior to tissue collection, the abalone were anesthetized with 5% MgCl 2 , after which tissue samples were collected. All collected tissues were washed with 1× PBS, immediately flash frozen in liquid nitrogen, and stored at −80 • C until required for total RNA extraction.

Collection of Ganglion Tissues of Pacific Abalone during Gonadal Development
Different ganglion tissues (CG, PPG, BG) of Pacific abalone were collected during different gonadal developmental stages. The gonadal developmental stages of Pacific abalone were determined as previously described [28]. The following developmental stages were evaluated in this study: immature stage (IM), developing stage (DS), ripen stage (RS), and spent stage (SS).

Collection of Ganglion Tissues of Pacific Abalone Conditioned at Effective Accumulative Temperature (EAT)
Two-year-old adult Pacific abalone of both sexes were collected from abalone sea-cage farm from the coast of Jindo-gun, Korea, during early December. All of the collected abalone were at the gonadal recovery phase. After collection, the abalone were transported to the Tou-Jeong Soosan Abalone Hatchery in Dolsan-eup and acclimatized for one week in rearing tanks with continuous aeration and running sea water. To induce early gonadal maturation of abalone, the broodstock is typically conditioned in abalone hatcheries using an effective accumulative temperature (EAT) at 18 • C for approximately 4 months to reach EAT of 1500 • C-days. In the present study, the abalone broodstock were conditioned under EAT exposer as previously described [23]. After acclimatizing at the natural water temperature (7 • ± 1 • C), the abalone were kept in an EAT-controlled rearing tank. The water temperature of the tank was steadily increased 1 • C at every alternative day from 7 • C to 18 • C, and finally maintained at 18 • C until the end of the broodstock management period. Ganglion tissues (CG, PPG, BG) were collected at 0 • C-days (i.e., the first day of condition), 500 • C-days, 1000 • C-days, and 1500 • C-days. Five abalones of each sex were sacrificed on each sampling day.

Collection of Ganglion of Pacific Abalone during Induced Spawning Events
Different ganglia (CG, PPG, BG) were collected from abalone at various steps of induced spawning events including heat induction (HI), induction with UV-irradiated water (UV), during spawning (DSW), and post-spawning (PSW). UV-and heat-induced spawning was performed using ripen stage abalone as previously described [23]. Briefly, the abalone at the gonadal ripen stage were first placed under direct sunlight at shelldown orientation for 1 h and shell-up orientation for half an hour. The abalone were then transferred to a spawning tank and induced with UV-irradiated seawater with moderate aeration. Five randomly selected abalone of both sexes were sacrificed at each stage of induced spawning events and ganglion tissues were collected. Finally, after 24 h of spawning, post-spawning samples were collected.

Collection of Different Embryonic and Larval Developmental Stages Samples of Pacific Abalone
After spawning induction, sperm and eggs were collected and mixed carefully in a 30 L bowl with filtered seawater (FSW) to promote successful fertilization. The water temperature of the bowl was maintained at 20 • C. The fertilized eggs were then washed three times with FSW (20 • C), after which the metamorphosis was undergo through different embryonic and larval stages. Samples were collected at the following embryonic and larval developmental stages: fertilized egg (FEG), morula (MOR), blastula (BLU), gastrula (GAS), trochophore larvae (TRL), early veliger larvae (EVL), operculate veliger larvae (OVL), late veliger larvae (LVL), pre-settlement larvae (PSL), and post larvae (PLV).

RNA Extraction and cDNA Synthesis
Total cellular RNAs were extracted from all collected tissues using the ISOSPIN Cell & Tissue RNA kit (Nippon Gene, Tokyo, Japan). First-strand cDNAs were synthesized from 1-4 µL of total RNA depending on the concentration of the total RNA using Superscript III First-strand cDNA synthesis kit (Invitrogen, USA). RACE (5 -and 3 -) cDNAs were synthesized from the total RNA of the CG using the SMARTer ® RACE 5 /3 Kit (Takara Bio Inc., Shiga, Japan). RNA extraction and cDNA synthesis were performed following the protocols provided by the manufacturer of the aforementioned kits.
2.6. Cloning and Sequencing of the Full-length FMRF2 Gene (Hdh-FMRF2) in Pacific Abalone 2.6.1. Cloning of Partial Sequence To obtain a partial fragment of Hdh-FMRF2 gene, reverse transcription polymerase chain reaction (RT-PCR) was performed using a cDNA template obtained from the CG, forward and reverse primers (Supplementary Table S1), and Phusion ® High-Fidelity DNA Polymerase (New England Biolabs Inc., Ipswich, MA, USA). The primers were designed from the known FMRF2 cDNA sequence of Haliotis asinina (GenBank Accession No. ACD65488). The cloning and sequencing of the partial fragment of the FMRF2 nucleotide was conducted using the TOPcloner™ Blunt kit (Enzynomics, Seoul, Republic of Korea) contains pTOP Blunt V2 vector and DH5α competent cells as previously described [28]. The thermal cycling conditions of the RT-PCR were the following: initial denaturation at 95 • C for 3 min; followed by 36 cycles of denaturation at 95 • C for 30 s, annealing at 58 • C for 1 min, and extension at 72 • C for 45 s; and a final extension step at 72 • C for 7 min. Sequencing was carried out at Macrogen (Seoul, Republic of Korea).

Cloning of 5 -and 3 -RACE Sequence
To obtain the full-length sequence of Hdh-FMRF2, rapid amplification of cDNA ends (RACE) PCR was performed in both the 5 -and 3 directions. Cloning and sequencing of 5 -and 3 -RACE fragments of Hdh-FMRF2 were performed using the SMARTer ® RACE 5 /3 kit (Takara Bio Inc., Shiga, Japan) as previously described [28]. Gene-specific 5and 3 -RACE primers (Supplementary Table S1) were designed from cloned Hdh-FMRF2 partial sequence following the recommendations of the SMARTer ® RACE 5 /3 kit. The thermal cycle conditions were also established according to the recommendations of the manufacturer of the kit. Finally, the sequences of the 5 -and 3 -RACE fragments were combined and the overlaps with the initially cloned partial sequence were trimmed to obtain the full-length sequence of Hdh-FMRF2.

Analysis of Amino Acid Sequence Alignment and Identity-Similarity Index
Representative amino acid sequences of FMRFamide were obtained from the NCBI protein database (https://www.ncbi.nlm.nih.gov/protein/; accessed on 25 July 2022). Amino acid sequences of FMRFamide from different animals were aligned using ClustalOmega (https://www.ebi.ac.uk/Tools/msa/clustalo/; accessed on 26 July 2022), an online multiple sequence alignment program. The aligned protein sequences were visualized and edited using the Jalview software v. 2.11.1.4. the amino acid sequence identity and similarity of Hdh-FMRF2 with those of other FMRFamide sequence were calculated using the "Idnt & Sim" online suite (https://www.bioinformatics.org/sms2/ident_sim.html; accessed on 10 August 2022). First, the amino acid sequences of the corresponding FMRFamide proteins were obtained from the NCBI protein database, aligned with the Uniprot online alignment tool (https://www.uniprot.org/align/; accessed on 10 August 2022), and finally analyzed with the "Idnt & Sim" suite against the Hdh-FMRF2 amino acid.

Phylogenetic Analysis
A phylogenetic tree was constructed using 54 FaRP protein sequences from different organisms obtained from the NCBI protein database. Phylogenetic analysis was conducted using the alignment build option of the MEGA software (v.11). First, the Hdh-FMFR2 amino acid sequence was aligned with other FaRP protein sequences using ClustalO (https: //www.ebi.ac.uk/Tools/msa/clustalo/; accessed on 2 August 2022). Then, a phylogenetic tree was constructed with the MEGA software (v. 11) using the neighbor-joining algorithm with 1000 bootstrap replicates. A detailed list of the FaRP sequences used for phylogenetic analysis is provided in Supplementary Table S3 centrifuged at 10,000× g for 10 min and supernatant were collected. Protein concentration was measured using Pierce TM BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Then, 100 µg protein was transferred in a micro tube and volume was adjusted to 100 µL of lysis solution. 50 µL of each reduction solution and alkylation solution were added to protein solution, mixed well and incubated at 95 • C for 10 min. After cooling, protein digestion was performed. To digest the protein, enzyme reconstitute solution and trypsin-protease mix was added to protein solution and incubated at 37 • C for 3 h in a shaking incubator. After incubation, the reaction was stopped by adding digestion stop solution. Later, peptide cleanup was performed using 300 µL digested protein and peptide clean-up column provided in the kit. The eluted peptide was dried by vacuum centrifugation in a speed-vac. The peptide was reconstituted with 100 µL 0.1% formic acid for MS/MS analysis. The peptide concentration was measured using Pierce TM quantitative fluorometric peptide assay kit (Thermo Fisher Scientific, Rockford, IL, USA).

Nano-LC-ESI-MS/MS and Peptide Identification
The Nano-LC-ESI-MS/MS analysis was performed using Quadrupole-Orbitrap instrument equipped with Dionex U 3000 RSLCnano HPLC system and Orbitrap Exploris 240 mass spectrometer (Thermo Fisher Scientific, Rockford, IL, USA). Peptide samples were pooled, and peptide fractionation was performed using HPLC system. Peptide fractions were then reconstituted in solvent A (Water/Acetonitrile 98:2 v/v, 0.1 % Formic acid) and then injected into LC-nano ESI-MS/MS system. Samples were first trapped onan Acclaim PepMap 100 trap column (100 µm × 2 cm, nanoViper C18, 5 µm, 100 Å) and washed with 98% solvent A at a flow rate of 4 µL/min for 6 min, and then separated on a PepMap RSLC C18 column (75 µm × 15 cm, nanoViper C18, 3 µm, 100 Å) at a flow rate of 300 nL/min. The LC gradient was run at 2% to 8% solvent B over 10 min, then from 8% to 30% over 55 min, followed by 90% solvent B for 4 min, and finally 2% solvent B for 20 min. Xcaliber software ver. 4.4 was used to collect MS data. The Orbitrap analyzer scanned the precursor ions with a mass range of 350-1800 m/z with 60,000 resolutions at m/z 200. Mass data are acquired automatically using proteome discoverer 2.5 (Thermo Fisher Scientific, Rockford, IL, USA). Fluorescence antisense and sense riboprobes were synthesized following a previously described protocol [29] with slight modifications. The mRNA probes were prepared from plasmid DNA of 626 bp fragments of the Hdh-FMRF2 cDNA that was subcloned into the pGEM-T easy vector (Promega, Madison, WI, USA). First, Hdh-FMRF2 plasmid DNA was linearized using 10 µg of plasmid DNA with the ApaI or SpeI restriction enzymes (Promega, USA) to obtain the antisense and sense probes, respectively. Then, the antisense and sense probes were separately labeled with fluorescein-12-UTP (Roche, Mannheim, Germany) using the T7 or SP6 RNA polymerases (Promega, Madison, WI, USA). The procedures were conducted using 20 µL of reaction mixture containing 1 µg of the linearized plasmid DNA, 4.0 µL of 5× optimized transcription buffer, 2.0 µL of 100 mM DTT, 2.0 µL of fluorescein-12-UTP labeling mix, 2.0 µL of RNase inhibitor, 2.0 µL T7 or SP6 RNA polymerase and RNasefree water (8.0 µL). The reaction mixture was incubated at 37 • C for 2 h. After incubation, the labeled linearized plasmid DNA template was digested at 37 • C for 15 min with DNase I (2.0 µL). The obtained riboprobes were then purified through ethanol precipitation with 1 µL of yeast tRNA (10 mg/mL). The purified riboprobes were stored at −80 • C until required for FISH.

Fluorescence in Situ Hybridization (FISH)
FISH were conducted according to the DIG fluorescein in situ hybridization application manual and a standard protocol previously described [30] with slight modifications. Hybridization buffer was prepared with 25 mL of deionized formamide, 12.5 mL of 20× saline sodium citrate (SSC), 0.5 mL of 10% Tween-20, 0.46 mL of 1M citric acid, and 11.44 mL of DEPC-H 2 O in a total volume of 50 mL. Afterward, hybridization buffer mix was prepared with yeast tRNA at a 9:1 ratio. Cryosections of CG tissue were first air dried for approximately 15 min, then washed with 1× PBS, and prehybridized with hybridization buffer mix at 65 • C for 2 h. After prehybridization, the sections were hybridized overnight with fluorescein RNA probe (300 ng/µL) at 65 • C. The tissue sections were then washed with a hybridization buffer gradient (75%, 50%, 25% volume) mixed with 2× SSC for 10 min each at 65 • C. The sections were then washed with 2× SSC and 0.2× SSC for 15 min each. Next, the sections were washed with a 0.2× SSC gradient (75%, 50%, 25% volume) mixed with PBST (5 min per wash) at room temperature. A final wash was performed with PBST for 5 min at room temperature. After the final wash, the sections were incubated at room temperature with 10% calf serum for 1 h. To detect the hybridization signal, the tissue sections were incubated at room temperature with Anti-Digoxigenin-Fluorescein Fab Fragments antibody (diluted at a 1:500 ratio in 10% calf serum) for 1 h. The tissue sections were then washed with PBST three times at room temperature for 10 min each. Finally, the hybridized sections were counterstained and mounted with VECTASHIELD antifade mounting medium with DAPI (4,6 -diamidino-2-phenylindole) (Vector Laboratories, Inc., Newark, CA, USA). Fluorescent mRNA signals were visualized and captured using a ZEISS LSM 900 confocal microscope with Airyscan2 (ZEISS, Oberkochen, Germany).

Quantitative Real-Time PCR (qRT-PCR) Analysis
The qRT-PCR analysis was performed to quantify the relative mRNA expression of Hdh-FMRF2 in different experimental tissues. Hdh-FMRF2 mRNA expression levels were quantified in 11 different tissues (CG, PPG, BG, HRT, OV, TE, DG, HCY, GIL, MNT, and MUS), CG at different gonadal developmental stages (DS, AS, RS, and SS), CG at different EAT • C-days (0, 500, 1000 and 1500 • C-days), CG at induced spawning events, and at different embryonic and larval developmental stages.
The qRT-PCR assay was conducted using the 2× qPCRBIO SyGreen Mix Lo-ROX kit (PCR Biosystems Ltd., London, UK) as previously described [31]. A 20 µL of qRT-PCR reaction mixture was prepared using 1 µL of cDNA template, 1 µL of each gene specific forward and reverse primer, 10 µL of SyGreen Mix, and 7 µL of DDW. Both the target and reference genes were analyzed in triplicate. The PCR conditions were the following: preincubation at 95 • C for 3 min, 40 cycles at 95 • C for 15 s, 60 • C for 20 s, and 72 • C for 20 s. The melting temperature conditions were the following: 95 • C for 10 s, 65 • C for 60 s, and 97 • C for 1 s. At the end of each cycle, a fluorescence reading was recorded for quantification. Florescence amplification and data analysis were performed using a LightCycler ® 96 System (Roche, Mannheim, Germany). Relative gene expression was calculated using the 2 −∆∆CT method using the β-actin gene of H. discus hannai as an internal reference. The expression level in the branchial ganglion was used as a reference value. Specifically, the expression level of the BG was set to 1 and all other data were normalized to this value. All primers used in qRT-PCR analyses are summarized in Supplementary Table S1. The qPCR primers of Hdh-FMRF2 were designed from the unmatched region from nucleotide sequence of Hdh-FMRF1, unmatched region was determined by sequence alignment of Hdh-FMRF2 and Hdh-FMRF1 using an online multiple sequence alignment program, ClustalW (https://www.genome.jp/tools-bin/clustalw; accessed on 27 July 2022).

Statistical Analysis
The values of mRNA expression of Hdh-FMRF2 in different organs, CG at different gonadal developmental stages, CG at different EAT • C-days, CG at different steps of spawning events, and different embryonic and larval developmental stages were expressed as mean ± standard error of the mean (SEM). The differences in the relative mRNA expression in different tissues were statistically assessed via nonparametric one-way analysis of variance (ANOVA) followed by Tukey's post hoc test using the GraphPad Prism 9.3.1 software. Statistical significance was assessed at p < 0.05. All graphs were also prepared using the GraphPad Prism 9.3.1 software. Different letters on the bar in the figures indicate significant differences (p < 0.05).

Haliotis discus hannai FMRFamide 2 (Hdh-FMRF2) Sequence
A full-length FMRFamide-related neuropeptide was cloned and sequenced from the CG tissue of Pacific abalone. The cloned FMRFamide-related peptide was termed as H. discus hannai FMRFamide 2 (Hdh-FMRF2) in this manuscript. The full-length Hdh-FMRF2 cDNA sequence (GenBank Accession No. MZ224009) was 1256 bp with a poly-A tail. The 5 -and 3 -untranslated regions (UTR) were 133 bp and 508 bp long, respectively ( Figure 1). A putative polyadenylation signal (AATAAA) was located at 12 bp upstream of the poly-A tail in its nucleotide sequence. The open reading frame (ORF) of the Hdh-FMRF2 cDNA sequence was 615 bp, encoding a putative protein of 204 predicted amino acids (NCBI Protein ID: QXP00688). Based on all of these features, a Hdh-FMRF2 protein sequence structure is presented in Figure 2A. Multiple sequence alignment analysis demonstrated that three cysteine residues were conserved at positions 20, 28, and 37 aa of the Hdh-FMRF2 amino acid sequence when aligned with other FMRFamide proteins of H. asinina and H. discus hannai ( Figure  2B). Among the compared invertebrate FMRFamide-related peptide sequences, Hdh-FMRF2 showed the highest identity (85.02) and similarity (89.86) with FMRF2 of H. asinina (Table 1). It also showed 28.96% and 37.01% identity and similarity, respectively, to both

Features of Hdh-FMRF2 Amino Acid Sequence and Bioinformatic Analysis
The theoretical molecular weight and isoelectric point (pI) of the Hdh-FMRF2 protein were 23.77 kDa and 9.7, respectively. The aliphatic index of the protein was 68.33. The instability index (II) was computed as 47.60, which classified the protein as unstable. The neural nets-nuclear prediction and integral prediction of protein location scores were 1.9 and 9.3, respectively, which predicted the cloned protein as an extracellular (secreted) protein.
Predicted GO term analysis using the C-I-TASSER server predicted that the Hdh-FMRF2 protein was significantly linked to the "neuropeptide hormone activity" term (GO:0005184) in the molecular function category with a C-score GO Figure 2A.
Multiple sequence alignment analysis demonstrated that three cysteine residues were conserved at positions 20, 28, and 37 aa of the Hdh-FMRF2 amino acid sequence when aligned with other FMRFamide proteins of H. asinina and H. discus hannai ( Figure 2B). Among the compared invertebrate FMRFamide-related peptide sequences, Hdh-FMRF2 showed the highest identity (85.02) and similarity (89.86) with FMRF2 of H. asinina (Table 1). It also showed 28.96% and 37.01% identity and similarity, respectively, to both FMRF1 of H. discus hannai and H. asinina (Table 1). Identity and similarity index showed that Hdh-FMRF2 and Hdh-FMRF1 have clear differences among their sequences. Table 2 provides a comparative summary of the characteristics of the predicted peptide sequences of Hdh-FMRF2 and Hdh-FMRF1. Peptide prediction clearly showed that there have distinct differences in peptide content among Hdh-FMRF2 and Hdh-FMRF1. A comparative summary of the characteristics of the predicted peptide sequences of Hdh-FMRF2 and Has-FMRF2 is also presented in Supplementary Table S2. that Hdh-FMRF2 and Hdh-FMRF1 have clear differences among their sequences. Table 2 provides a comparative summary of the characteristics of the predicted peptide sequences of Hdh-FMRF2 and Hdh-FMRF1. Peptide prediction clearly showed that there have distinct differences in peptide content among Hdh-FMRF2 and Hdh-FMRF1. A comparative summary of the characteristics of the predicted peptide sequences of Hdh-FMRF2 and Has-FMRF2 is also presented in Supplementary Table S2.    The motifs of FMRFamide were parallelly expressed among the compared FMRFamide protein sequences. The motif widths were within 11 and 50 amino acids. Five motifs were identified in Hdh-FMRF2. Likewise, five motifs were identified in the Has-FMRF2 protein, whereas seven motifs were detected in Hdh-FMRF1 and Has-FMRF1 proteins. Five motifs were also recognized in B. glabrata among which three motifs matched with Hdh-FMRF2 ( Figure 3). The motifs of FMRFamide were parallelly expressed among the compared FMR-Famide protein sequences. The motif widths were within 11 and 50 amino acids. Five motifs were identified in Hdh-FMRF2. Likewise, five motifs were identified in the Has-FMRF2 protein, whereas seven motifs were detected in Hdh-FMRF1 and Has-FMRF1 proteins. Five motifs were also recognized in B. glabrata among which three motifs matched with Hdh-FMRF2 (Figure 3).

Three-Dimensional Structure and Pairwise 3D Structure Alignment
The 3D structure of the FMRF2 protein in H. discus hannai and a pairwise 3D structure alignment of FMRF2 protein of H. discus hannai and H. asinina are presented in Figure 4A and 4B, respectively. The 3D structures of Hdh-FMRF2 were characterized by several alpha helices separated by loops, forming a helix-loop-helix (HLH) structure. The 3D structure of Hdh-FMRF2 characterized by eight alpha helices was separated by seven loops ( Figure 4A) with a C-score of −2.56, an estimated TM-score of 0.42 ± 0.14, and an estimated root-mean-square deviation (RMSD) of 11.3 ± 4.6Å. The pairwise 3D structure alignment of Hdh-FMRF2 and Has-FMRF2 showed a close structural relation ( Figure 4B).

Phylogenetic Analysis
To assess the potential evolutionary connections of Hdh-FMRF2 with other FaRPs from vertebrates, annelids, arthropods, and mollusks, a phylogenetic tree was constructed using the neighbor-joining method. The unrooted phylogenetic tree exhibited two major clades, invertebrate FaRP and vertebrate FaRP; invertebrate FaRP further sub-clustered in the invertebrate FLRP and invertebrate FMRF ( Figure 5). Hdh-FMRF2 was fitted in the invertebrate FMRFamide clade and sub-grouped with gastropod species. Moreover, Hdh-FMRF2 was fitted with its closest phylogenetic matches, FMRF2 of H. asinina. alignment of FMRF2 protein of H. discus hannai and H. asinina are presented in Figure 4A and 4B, respectively. The 3D structures of Hdh-FMRF2 were characterized by several alpha helices separated by loops, forming a helix-loop-helix (HLH) structure. The 3D structure of Hdh-FMRF2 characterized by eight alpha helices was separated by seven loops ( Figure 4A) with a C-score of -2.56, an estimated TM-score of 0.42±0.14, and an estimated root-mean-square deviation (RMSD) of 11.3 ± 4.6Å. The pairwise 3D structure alignment of Hdh-FMRF2 and Has-FMRF2 showed a close structural relation ( Figure 4B).

Phylogenetic Analysis
To assess the potential evolutionary connections of Hdh-FMRF2 with other FaRPs from vertebrates, annelids, arthropods, and mollusks, a phylogenetic tree was constructed using the neighbor-joining method. The unrooted phylogenetic tree exhibited two major clades, invertebrate FaRP and vertebrate FaRP; invertebrate FaRP further sub-clustered in the invertebrate FLRP and invertebrate FMRF ( Figure 5). Hdh-FMRF2 was fitted in the invertebrate FMRFamide clade and sub-grouped with gastropod species. Moreover, Hdh-FMRF2 was fitted with its closest phylogenetic matches, FMRF2 of H. asinina.

Identification of FaRP Peptides
CG tissues were examined to detect the presence of mature FaRPs. Representative Nano-LC-ESI-MS/MS clearly showed mass ion peak corresponding to FMRFamide and YLRFamide. Further, NFGEPFLRFa, FGRNFGEPFLRFa, FDSYEDKAYLRFa, SDPGEDP MLKAILLRGAPNNNGWQY and NGWLHFa peptide peaks were also detected (Table 3 and Supplementary Figure S2).

Fluorescence in Situ Hybridization (FISH) Localization of Hdh-FMRF2
FISH analyses revealed that the Hdh-FMRF2 riboprobe showed positive signals in neurosecretory cell bodies of the cortex region in the CG when hybridized with the fluores-cence antisense probe ( Figure 6). In contrast, hybridization with fluorescence sense probe of Hdh-FMRF2 did not show any signal ( Figure 6). These data indicate that Hdh-FMRF2 mRNA is exclusively expressed in cell bodies in the cortex of the CG in Pacific abalone.

Expression Levels of Hdh-FMRF2 mRNA in Different Tissues of Pacific Abalone
The relative mRNA expression levels of Hdh-FMRF2 in various tissues of Pacific abalone were analyzed by qRT-PCR and the results are presented in Figure 7. The Hdh-FMRF2 gene expression was not only exclusively expressed in CG, a lower level of expression was also found in other organs. A negligible expression of Hdh-FMRF2 was detected in the GIL, DG, MNT, MUS, and HCY. Moreover, the expression levels among male and female were not significantly different.

Expression Levels of Hdh-FMRF2 in CG during Gonadal Development of Pacific Abalone
At different gonadal developmental stages, changes in relative mRNA expression of Hdh-FMRF2 were observed in the CG of male and female abalone. The expression of Hdh-FMRF2 was found to be significantly higher in the RS in both sexes (Figure 8). The expression levels of Hhd-FMRF2 were markedly increased from IM to DS and DS to RS but decreased in SS, and the expression levels among developmental stages were significantly different. A lower mRNA expression of Hdh-FMRF2 was observed in female at the ripen stage compared to male, however, these changes were statistically insignificant.

Expression Levels of Hdh-FMRF2 in CG during Gonadal Development of Pacific Abalone
The mRNA expression levels of Hdh-FMRF2 in the CG of EAT conditioned abalone were significantly higher at EAT 1500 °C-days, when the abalone were fully mature (Figure 9). The expression of Hdh-FMRF2 was significantly increased from EAT 500 °C-days to EAT 1000 °C-days and from EAT 1000 °C-days to EAT 1500 °C-days with the progress of gonadal maturation. However, the differences in mRNA expression among EAT 0 °Cdays and EAT 500 °C-days were insignificant when the gonads were in the inactive stage.

Expression Levels of Hdh-FMRF2 in CG during Gonadal Development of Pacific Abalone
The mRNA expression levels of Hdh-FMRF2 in the CG of EAT conditioned abalone were significantly higher at EAT 1500 • C-days, when the abalone were fully mature (Figure 9). The expression of Hdh-FMRF2 was significantly increased from EAT 500 • C-days to EAT 1000 • C-days and from EAT 1000 • C-days to EAT 1500 • C-days with the progress of gonadal maturation. However, the differences in mRNA expression among EAT 0 • C-days and EAT 500 • C-days were insignificant when the gonads were in the inactive stage.
The mRNA expression levels of Hdh-FMRF2 in the CG of EAT conditioned abalone were significantly higher at EAT 1500 °C-days, when the abalone were fully mature (Figure 9). The expression of Hdh-FMRF2 was significantly increased from EAT 500 °C-days to EAT 1000 °C-days and from EAT 1000 °C-days to EAT 1500 °C-days with the progress of gonadal maturation. However, the differences in mRNA expression among EAT 0 °Cdays and EAT 500 °C-days were insignificant when the gonads were in the inactive stage. Figure 9. Changes in mRNA expression levels of Hdh-FMRF2 in the cerebral ganglion of different EAT °C-days of Pacific abalone in both sexes exposed to EAT during broodstock conditioning. For all bar graphs, the raw data points (green circle) represent biological replicates, the error bars represent the standard error of the mean (SEM), and different letters above the bars indicate significant differences (p < 0.05) among EAT °C-days. EAT 00, EAT 0°C-days; EAT 500, EAT 500°C-days; EAT 1000, EAT 1000°C-days; EAT 1500, EAT 1500°C-days. Figure 9. Changes in mRNA expression levels of Hdh-FMRF2 in the cerebral ganglion of different EAT • C-days of Pacific abalone in both sexes exposed to EAT during broodstock conditioning. For all bar graphs, the raw data points (green circle) represent biological replicates, the error bars represent the standard error of the mean (SEM), and different letters above the bars indicate significant differences (p < 0.05) among EAT • C-days. EAT 00, EAT 0 • C-days; EAT 500, EAT 500 • C-days; EAT 1000, EAT 1000 • C-days; EAT 1500, EAT 1500 • C-days.

Expression Levels of Hdh-FMRF2 mRNA in CG during Induced Spawning Events
During induced spawning events, the mRNA expression levels of Hdh-FMRF2 were significantly higher at the spawning stage (DSW) and they were significantly downregulated at PSW (Figure 10). Moreover, although a gradual increase in the mRNA expression of Hdh-FMRF2 was observed among IC, HI and UV stages, the changes were not statistically significant. Interestingly, the mRNA expression levels in the female at the DSW stage were significantly different compared to those of the male. The changes in the mRNA expression levels between the UV stage and DSW stage in the female were not significant, whereas these changes were significant in the male.

Expression Levels of Hdh-FMRF2 mRNA in CG during Induced Spawning Events
During induced spawning events, the mRNA expression levels of Hdh-FMRF2 were significantly higher at the spawning stage (DSW) and they were significantly downregulated at PSW (Figure 10). Moreover, although a gradual increase in the mRNA expression of Hdh-FMRF2 was observed among IC, HI and UV stages, the changes were not statistically significant. Interestingly, the mRNA expression levels in the female at the DSW stage were significantly different compared to those of the male. The changes in the mRNA expression levels between the UV stage and DSW stage in the female were not significant, whereas these changes were significant in the male. For all bar graphs, the raw data points (green circle) represent biological replicates, the error bars represent the standard error of the mean (SEM), and different letters above the bars indicate significant differences (p < 0.05) among the steps of induced spawning event. IC, initial control; HI, heat induced; UV, UV-irradiated water induced; DS, during spawning, AS, after spawning.

Expression Levels of Hdh-FMRF2 mRNA in Embryonic and Larval Developmental Stages of Pacific Abalone
The mRNA expression of Hdh-FMRF2 was first detected in trochophore larvae (TRL) at 16 h post-fertilization (hpf). Later, it was significantly increased in the EVL stage at 24 hpf. However, the expression levels gradually increased until post-larval stage (PLV) at 144 hpf and significantly higher expression levels were observed at PLV (Figure 11). No mRNA expression was observed in any of the examined embryonic stages. Figure 10. Changes in mRNA expression levels of Hdh-FMRF2 in the cerebral ganglion during different steps of induced spawning events of Pacific abalone in both sexes. For all bar graphs, the raw data points (green circle) represent biological replicates, the error bars represent the standard error of the mean (SEM), and different letters above the bars indicate significant differences (p < 0.05) among the steps of induced spawning event. IC, initial control; HI, heat induced; UV, UV-irradiated water induced; DS, during spawning, AS, after spawning.

Expression Levels of Hdh-FMRF2 mRNA in Embryonic and Larval Developmental Stages of Pacific Abalone
The mRNA expression of Hdh-FMRF2 was first detected in trochophore larvae (TRL) at 16 h post-fertilization (hpf). Later, it was significantly increased in the EVL stage at 24 hpf. However, the expression levels gradually increased until post-larval stage (PLV) at 144 hpf and significantly higher expression levels were observed at PLV (Figure 11). No mRNA expression was observed in any of the examined embryonic stages.

Discussion
FMRFamide-related neuropeptides (FaRPs) are important regulators of reproductive regulation, feeding behavior, larval development, and central nervous system development in mollusk species [32]. FaRPs have been reported in all classes of mollusk species, including gastropods, cephalopods, and bivalve mollusks. In the present study, a transcript of FMRFamide gene (Hdh-FMRF2) was cloned from Pacific abalone, a gastropod mollusk species. After cloning, it was sought to determine whether this gene is involved in the reproductive regulation and larval development of Pacific abalone.
In this study, a full-length cDNA of H. discus hannai FMRFamid 2 (Hdh-FMRF2) was sequenced and characterized from the CG of Pacific abalone. Hdh-FMRF2 protein contains four copies of FaRP domain (two copies of FLRFa, one copy of YLRFa and one copy of FMRFa). To designate a peptide as a true FaRP, the following C-terminal sequence is required: an aromatic amino acid, such as phenylalanine (F), tyrosine (Y), or tryptophan (W); an amino acid with a hydrophobic group such as phenylalanine (F), isoleucine (I), leucine (L), methionine (M), threonine (T) or valine (V); and a C-terminal arginine (R) and phenylalanine (F) amide (NH2) [18]. The four FaRP domains characterized in the Hdh-FMRF2 met the aforementioned conditions and therefore Hdh-FMRF2 could be classified as a true FaRP. This peptide also contained a highly conserved tetrabasic furin-processing site (RRKR) that separated the peptide into N-terminal and C-terminal region. Previously, an FMRFamide peptide had been reported in Pacific abalone, which was designated as FMRF1 (GenBank Accession no. AVW85483.1). The gene cloned in this study is the second transcript of an FMRFamide peptide isolated from the Pacific abalone, therefore, this peptide is designated as Hdh-FMRF2. Two transcripts of the FMRFamide peptide have also been reported in several mollusk species, including tropical abalone [2], marsh snail [33], European cuttlefish [34], and great pond snail [35].

Discussion
FMRFamide-related neuropeptides (FaRPs) are important regulators of reproductive regulation, feeding behavior, larval development, and central nervous system development in mollusk species [32]. FaRPs have been reported in all classes of mollusk species, including gastropods, cephalopods, and bivalve mollusks. In the present study, a transcript of FMRFamide gene (Hdh-FMRF2) was cloned from Pacific abalone, a gastropod mollusk species. After cloning, it was sought to determine whether this gene is involved in the reproductive regulation and larval development of Pacific abalone.
In this study, a full-length cDNA of H. discus hannai FMRFamid 2 (Hdh-FMRF2) was sequenced and characterized from the CG of Pacific abalone. Hdh-FMRF2 protein contains four copies of FaRP domain (two copies of FLRFa, one copy of YLRFa and one copy of FMRFa). To designate a peptide as a true FaRP, the following C-terminal sequence is required: an aromatic amino acid, such as phenylalanine (F), tyrosine (Y), or tryptophan (W); an amino acid with a hydrophobic group such as phenylalanine (F), isoleucine (I), leucine (L), methionine (M), threonine (T) or valine (V); and a C-terminal arginine (R) and phenylalanine (F) amide (NH 2 ) [18]. The four FaRP domains characterized in the Hdh-FMRF2 met the aforementioned conditions and therefore Hdh-FMRF2 could be classified as a true FaRP. This peptide also contained a highly conserved tetrabasic furin-processing site (RRKR) that separated the peptide into N-terminal and C-terminal region. Previously, an FMRFamide peptide had been reported in Pacific abalone, which was designated as FMRF1 (GenBank Accession no. AVW85483.1). The gene cloned in this study is the second transcript of an FMRFamide peptide isolated from the Pacific abalone, therefore, this peptide is designated as Hdh-FMRF2. Two transcripts of the FMRFamide peptide have also been reported in several mollusk species, including tropical abalone [2], marsh snail [33], European cuttlefish [34], and great pond snail [35].
Phylogenetic tree analyses revealed two FaRP clades representing invertebrate and Vertebrate FaRPs. The invertebrate FaRP clade was further sub-clustered into the invertebrate FMRFamide subfamily and the invertebrate LFRFamide subfamily. As expected, the organization of the invertebrate FaRPs in a separate clade in the phylogenetic tree supported their evolutionary relationship. The Hdh-FMRF2 was fitted in the invertebrate FaRP clade and was grouped with almost all mollusk species. It subclustered with its closest relative gastropod mollusk species, H. asinina and Gigantopelta aegis, revealing that this neuropeptide has a tight evolutionary relationship with gastropod species.
Fluorescence in situ hybridization localization in adult Pacific abalone revealed that the Hdh-FMRF2 mRNA antisense probe expressed positive signals in the neurosecretory cell bodies in the CG. In situ hybridization and immunohistochemical localization of FMRFamide in adult ganglion tissues and the developing central nervous system have been reported in several mollusk species including tropical abalone [2], common Chinese cuttlefish [13], longfin squid [15], pygmy squid [36] and common octopus [37]. With the analysis of the distribution of RFamide peptide in the central nervous system of mollusks, it has been demonstrated that RFamide peptides localized in the cerebral ganglia are involved in reproduction [14].
The tissue distribution analysis using qRT-PCR revealed that Hdh-FMRF2 showed significantly higher mRNA expression in the CG of male and female abalone. Although female expressed lower mRNA levels of Hdh-FMRF2 than the male, these differences were insignificant. Furthermore, a considerable level of expression was observed in the PPG (a ganglion connected to the CG), the heart and the gonads. Negligible levels of mRNA expression were also observed in other tested tissues. In addition to the central nervous system, FMRFamide peptides have been reported in various organs of different mollusk species, such as in the heart of sunray venus clam [5], common Chinese cuttlefish [13], and sea hare [38] as well as in the reproductive organs of common Chinese cuttlefish [13] and garden snail [39].
Hdh-FMRF2 mRNA expressions were detected in the CG of all stages of gonadal development. The expression was progressively upregulated from the IM to the RS stages, but it was downregulated at the SS. A similar expression pattern has also been reported in the brain of pharaoh cuttlefish [7]. Hdh-FMRF2 expression was significantly higher at the RS in both sexes. Female abalone exhibited lower expression levels than male; however, these differences were insignificant. The changes in mRNA expression between the DS and RS in female were also not significant. A similar mRNA expression pattern was observed in the CG of EAT-conditioned abalone as observed in the normal gonadal development process. The mRNA expression levels of Hdh-FMRF2 were increased gradually from EAT 0 • C-days (immature) to EAT 1500 • C-days (fully mature) with the progress of gonadal maturation. The mRNA expression levels reached in a peak at EAT 1500 • C-days. The female exhibited lower expression levels than the male; however, these differences were not significant. The changes in mRNA expression of Hdh-FMRF2 between EAT 1000 • C-days and EAT 1500 • C-days in female were also not significant. These insignificant changes were also observed during the normal gonadal developmental process of female between DS and RS. The involvement of FMRFamide or FaRP in gonadal development and reproduction has been reported in several invertebrate species. For example, differential expression has been reported in the brain of pharaoh cuttlefish among stage IV and stage V; and female showed significantly lower expression levels at stage V than the male, whereas the opposite trend was observed at stage IV [7]. FMRFamide immunoreactivity was also found in the reproductive ducts of octopus, suggesting that FMRFamide might participate in the central and peripheral peptidergic control of reproduction of common octopus [8]. Moreover, brain FMRFamide influences the secretory activity of the optic glands that control the maturation of the reproductive system in common octopus [40]. Previous studies have also demonstrated that FMRFamide might control ovulation, egg movement and oviposition in the kissing bug [41], as well as play a modulatory role in the locomotion and reproduction of roundworm [42]. Collectively, these findings suggest that Hdh-FMRF2 is a reproductionassociated peptide in abalone and is involved in the reproductive maturation process.
During induced spawning of Pacific abalone, the mRNA expression of Hdh-FMRF2 in the CG reached its peak at spawning time or during gamete release (DSW) compared to the other stages. However, female abalone showed significantly lower mRNA expression levels than male at the DSW stage. The changes in mRNA expression levels between the UV and DSW stages in the female were not significant, however, these changes were significant in the male. The mRNA expression levels were significantly downregulated at the PSW stage compared to DSW in both sexes. A previous study reported that expression of FMRFamide significantly decreased just prior to spawning in female tropical abalone, whereas the opposite was observed in the male [43]. The inhibitory effect of FMRFamide on oviposition has been demonstrated in several mollusk species including the great pond snail [44], sea hare [45] and tropical abalone [43]. Moreover, previous studies have confirmed that FMRFamide expression significantly decreased at the time of spawning in female tropical abalone compared to the male [43], which is consistent with the results of the present study. The subsequent downregulation or insignificant increase of FMRFamide during spawning time in females has also been observed in the great pond snail [44] and sea hare [45]. The present findings of lower expression of Hdh-FMRF2 during gamete release in female abalone compared to male may suggest that Hdh-FMRF2 might play an inhibitory role in the spawning or release of gametes in female Pacific abalone but not in the male. Similar to the findings of the present study, a previous study also demonstrated that the expression levels of FMRF2 significantly decreased at 24 h of post-spawning in tropical abalone [43]. However, for concrete evaluation, further experiments are needed on peptide quantification and peptide bioassay at spawning stage abalone.
The mRNA expression analysis of Hdh-FMRF2 during the embryonic and larval development of Pacific abalone revealed that this peptide was not expressed in any of the embryonic stages (fertilized egg, morula, blastula and gastrula). After hatching, the mRNA expression of Hdh-FMRF2 was detected in trace levels in trochophore larvae. However, Hdh-FMRF2 expression was significantly increased at the early veliger stage (EVL) when the nervous system began to develop. The expression levels subsequently increased until post-larval stage at which stage the highest expression of Hdh-FMRF2 was observed. Early studies reported that cerebral ganglia first appeared at the early veliger stage in European abalone [46]. Based on RT-PCR and ISH analyses, it has been reported that Has-FMRF2 expression was first detected at the trochophore larvae in tropical abalone at 12 hpf and expression was detected in all larval stages until 112 hpf [2]. The present results of the mRNA expression of Hdh-FMRF2 during the embryonic and larval development stages of Pacific abalone showed similar results as demonstrated in tropical abalone. Furthermore, in situ hybridization and immunohistochemical localization of FMRFamide in larval development stages have also been reported in several mollusk species including tropical abalone [2], Pacific oyster [47], nudibranch gastropod sea slug [48], Italian river snail [49] and sea hare [50]. Therefore, the results of the present study suggest that Hdh-FMRF2 plays a regulatory role in the larval development and nervous system development of Pacific abalone larvae.

Conclusions
Hdh-FMRF2, a second transcript of FMRFamide, was cloned from Pacific abalone. The mRNA expression analyses at different reproductive development experiments revealed that Hdh-FMRF2 was potentially involved in reproductive maturation. Furthermore, mRNA expression analyses in induced spawning event suggested that Hdh-FMRF2 inhibits gamete release in female abalone but not in the male. Hdh-FMRF2 neuronal cell bodies were predominantly localized in the CG. Furthermore, its mRNA expression was observed in all larval developmental stages from the beginning of the nervous system development but not in the preceding embryonic stages, suggesting that Hdh-FMRF2 regulates the larval development of Pacific abalone larvae. Collectively, the present findings provide important insights into the involvement of specific FaRP (Hdh-FMRF2) in reproductive maturation and larval development of Pacific abalone. The results of this study might be valuable for further studies on FaRPs in abalone and useful in broodstock management in abalone hatcheries and aquaculture as well.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biom13010109/s1, Figure S1: Gene ontology (GO) of Hdh-FMRF2.  Table S1: List of primers used for cDNA cloning, in situ hybridization, and qRT-PCR analysis of Hdh-FMRF2 in Pacific abalone. Table S2: Comparison of peptide predicted between Hdh-FMRF1 and Has-FRMF2 precursors. Table S3: Sequence information of FaRPs used for motif analysis, multiple sequence alignment and phylogenetic analysis.