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The Neuropeptide HGAP Regulates Growth, Reproduction, Metamorphosis, Tissue Damage Repair, and Response against Starvation in Pacific Abalone

The Neuropeptide HGAP Regulates Growth, Reproduction, Metamorphosis, Tissue Damage Repair, and... IntroductionNeuroendocrine systems play vital roles in regulating growth and reproduction by releasing amino acid derivatives, proteins, and peptides [1]. In mollusks, the neural ganglia secrete numerous neuropeptides that regulate brain activity in response to signals. The cerebral and pleuropedal ganglia are particularly important for producing neuropeptides involved in mollusk growth and reproduction [2, 3]. However, the activity of a neuropeptide may not be limited to growth and reproduction; it may exhibit diverse physiological functions in the organism.Growth of all living organisms is influenced by genetic, nutritional, and environmental factors [4]. In vertebrates, growth is primarily regulated by the growth hormone (GH) and insulin-like factor (IGF) axes, which are secreted by neuroendocrine cells [5]. Although GH has not been identified in invertebrates, insulin-like peptides and related derivatives are known to play key roles in various growth-related processes, including glucose, lipid, and amino acid metabolism in mollusks [6, 7]. In addition to GH and IGFs, species-specific growth factors and peptides contribute to growth regulation in different organisms.Neuroendocrine components control cell signal communication and play crucial roles in gamete formation, regulation, maturation, and release in both vertebrates and invertebrates [8]. Neural ganglia produce neuropeptides that act as reproductive regulators and participate in the reproductive process [9, 10]. The regulation of the reproductive cycle is attributed, at least in part, to a complex combination of neurohormones rather than a single peptide [11]. Detailed analyses of reproduction-related genes and their functions are being carried out in various mollusk species, such as freshwater snails, cuttlefish, and oysters, using transcriptomic resources [12‒16]. Gonadotropin-releasing hormone (GnRH) is a major neuroendocrine hormone that plays a central role in controlling reproductive process in abalone. While GnRH is a representative neuropeptide for regulating reproductive function in vertebrates and invertebrates, other neuropeptides such as caudo-dorsal cell hormone, FMRFamide, and APGWamide also play roles in various reproductive activities [17].In addition to these neuropeptides, there are some species-specific neuropeptides, such as molluscan insulin-related peptide (MIP) and haliotid growth-associated peptide (HGAP), which have been reported to regulate both growth and reproduction in mollusks [11, 18]. Among these, HGAP is specific to abalone. Previous research first identified the HGAP neuropeptide in Haliotis asinina, where it was found to be upregulated in the neural ganglia. In the context of abalone production, there is considerable interest in improving growth rates and controlling reproduction.Despite losses in abalone capture yields in natural environments, abalone aquaculture has experienced significant growth worldwide in recent decades [19, 20]. In South Korea, abalone aquaculture production reached 23,199 tons in 2021, accounting for 30% of the country’s shellfish production [21]. As abalone aquaculture relies entirely on hatchery-produced seed, understanding the reproductive behavior and regulation of abalone is crucial for successful seed production. One of the major challenges in abalone aquaculture, both in farm- and sea cage-based cultures, is achieving desirable growth rates. The growth and reproduction of abalone are genetically regulated by various hormones, peptides, and their derivatives related to growth and reproduction. While most of these molecules have specific functions in either growth or reproduction regulation, some may exhibit multifunctional roles. A recent transcriptome study on the ass’s-ear abalone H. asinina identified a specific neuropeptide called HGAP, which is reported to regulate both growth and reproduction of abalone. However, there have been no studies characterizing this neuropeptide and exploring its diverse regulatory roles in Pacific abalone. Considering that Pacific abalone (Haliotis discus hannai) is the most commercially cultured marine gastropod species in South Korea, this study aimed to clone, characterize, and investigate the regulatory role of HGAP in Pacific abalone. Particularly, this study will focus on various aspects such as growth, metamorphosis, muscle tissue remodeling, response to starvation, and reproduction, using expression analysis.MethodsExperimental AnimalsThree-year-old sexually mature Pacific abalones with an average body weight of 120.4 ± 0.61 g and an average shell length of 84.06 ± 0.32 mm were collected from sea cages in Jindo-gun, South Korea. After carefully observing the health status of abalones in different age groups, we have identified this three-year-old Pacific abalone as suitable for the present study. The abalones were transported using oxygenated plastic tanks to the Tou-Jeong Soosan abalone hatchery in Dolsan-eup, Yeosu, South Korea, where they were cultured for 1 month with sufficient food and water supply to recover from the transportation-related stress. After adjustment, these abalones were used for cloning and tissue-specific expression analysis, as well as for artificial breeding, GnRH peptide treatment, starvation treatment, and experiments related to muscle tissue remodeling.Tissue Collection for Gene Cloning and Expression AnalysisFor cloning and tissue-specific expression analysis, a total of 12 Pacific abalones were sacrificed, and their hemocytes (HMC), cerebral ganglion (CG), pleuropedal ganglion (PPG), testis (TES), ovary (OVR), mantle (MNT), muscle (MUS), gill (GIL), heart (HRT), and digestive gland (DG) tissues were collected. Prior to tissue sample collection, the abalones were anesthetized using 5% MgCl2. The collected tissue samples were washed with phosphate-buffered saline (PBS; 0.1 m), immediately snap-frozen in liquid nitrogen (LN2), and stored at −80°C until total RNA extraction.Embryo and Larvae CollectionThree-year-old reproductively mature 12 females’ and 4 male’s abalone were induced for artificial fertilization. After spawning, unfertilized eggs (UFE) and sperm were collected, and artificial fertilization was performed following the procedure described by Hanif et al. [22]. After fertilization, samples of target stages, including fertilized eggs (FE), 2-cell (2-CL) and 4-cell (4-CL) stages, morula (MOR), trochophore (TRP) larvae, veliger (VLG) larvae, and juveniles (JUV), were collected through microscopic observation. The juvenile sample was collected monthly for up to 6 months. The samples were immediately snap-frozen in LN2 and stored at −80°C until total RNA extraction.Juvenile Abalone Culture and Growth Type Sample CollectionAfter fertilization, settlement larvae were transferred to a culture tank with diatoms precoated on a transparent and flat plastic film. For successful settlement of larvae in the tank and plastic sheet, we provided enough aeration in the tank, but seawater supply was stopped on the first day of larvae release to avoid washout of larvae. The early juvenile abalones were fed diatoms on plastic films and provided artificial powder feed (composed of kelp powder, fish meal powder, micronutrients, and effective microorganisms) for 2 months. In South Korea, several artificial powder feeds and granular feeds of different sizes are available for abalone nursing at the farm level. Importantly, artificial feed has been shown to perform better than diets exclusively based on algae in abalone aquaculture. After 2 months, we switched from powder feed to granular feed (2 × 1 mm) for an additional 2 months. Next, 3 × 1 mm granular feed was provided for 1 year. After 6 months, juvenile abalones were collected and sorted according to size. Afterward, length, width, and weight data were taken, tagged, and again reared in the tank for 1 year. One year later, we collected all the abalones, measured them, and compared them with previous data. After comparison, we categorized them as follows: SG, stunted growth (size increased >3 mm); MG, minimal growth (size increased 5 mm to >1 cm); NG, normal growth (size increased 1–2 cm); and RG, rapid growth (size increased <2 cm). Then, muscle tissue samples of five juveniles from each category were collected. The samples were flash-frozen in LN2 and stored at −80°C until total RNA extraction.Tissue Sample from Injured Pacific AbaloneThree-year-old mature Pacific abalones (n = 40) with an average body weight of 121.2 ± 0.53 g and an average shell length of 84.11 ± 0.51 mm were used for the muscle tissue remodeling experiment. The abalones were divided into two groups where group 1 served as control and group 2 as for remodeling treatment. Briefly, 3 × 1 mm pieces of muscle tissue were carefully removed from 25 abalones using a tissue-collecting needle after anesthetizing them with 5% MgCl2. Subsequently, they were cultured in a rearing tank with continuous aeration, water, and a food supply. Muscle tissue samples from the injured region of three abalones were collected at four time points: 1 day, 6 days, 12 days, and 24 days. Additionally, tissue samples from control (initial) abalones and remodeled abalones (after 42 days of injury) were collected. All samples were washed with PBS, immediately snap-frozen in LN2, and stored at −80°C until total RNA extraction.Tissue Collection at Different Gonad Developmental StagesIn the reproductive season, samples of CG and gonad tissue (TES and OVR) were collected at different gonadal development stages, including the spent stage (SS), proliferative stage (PS), ripening stage (RS), and degenerative stage (DS). The tissue samples were immediately flash-frozen in LN2 and stored at −80°C until total RNA extraction.Administration of GnRH Peptide in the Gonad of Pacific AbaloneDuring the resting/spent stage (when gonad was completely empty), adult male (n = 20, body weight 123.43 ± 4.6 g, shell length 84.24 ± 2.6 mm) and female (n = 20, body weight 121.52 ± 4.9 g, shell length 83.94 ± 2.6 mm) were reared in the cemented tank with running sea water in the hatchery. Forty individuals were divided into four groups (two female groups and two male groups). Group 1 (male) and 3 (female) were considered control, treated with PBS. Group 2 (male) and 4 (female) were injected with a synthetic GnRH peptide (pQNYHFSNGWYA-NH2) of Pacific abalone (accession No. AZL93822.1), generated by ANYGEN (Gwangju, South Korea) at dose of 250 ng/g body weight into the pedal sinus of Pacific abalone. The water temperature was maintained at 22 ± 0.6°C during the experiment. The CG, TES, and OVR samples from each group were randomly collected from five individuals after 15 days, 30 days, and 45 days. All samples were washed with PBS, immediately snap-frozen in LN2, and stored at −80°C until total RNA extraction.Tissue Samples from Starved Pacific AbalonesThree-year-old Pacific abalone (n = 30, shell length 84.43 ± 0.62 mm; body mass 121.7 ± 3.36 g) were randomly collected from the sea cages in Wando-gun, South Korea, and transported to the abalone hatchery in Yeosu, South Korea. The collected abalones were reared in tanks with running seawater flow, aeration, and adequate feed supply for adjustment. The abalones were divided into two groups, where group A served as control and group B for starvation treatment. At the beginning of starvation treatment, muscle tissue from three control individuals was collected. Since then, group B abalones were reared without feeding, and muscle tissue was collected from three abalones at 1-week intervals for 3 weeks. Afterward, starved abalones were reared with feed for 1 day, and samples were collected. Samples from control abalones were also collected at each sampling period following the procedure mentioned above and stored at −80°C until total RNA extraction.Total RNA Extraction and First-Stand cDNA SynthesisTotal cellular RNA was extracted from the collected tissue samples using the ISOSPIN Cell and Tissue RNA Kit (Nippon Gene, Tokyo, Japan). First-strand cDNA synthesis was performed using extracted RNA, oligo(dt) primers (Sigma), and the Superscript III First-Strand cDNA Synthesis Kit (Invitrogen, USA) (Table 1). The RACE cDNA (3′- and 5′-RACE) was synthesized from the extracted total RNA using the SMARTer® RACE 5′/3′ Kit (Takara Bio Inc., Japan). Both total RNA extraction and cDNA synthesis were conducted according to the manufacturer’s instructions.Table 1.List of different primers used for cDNA synthesis, cloning, and expression analysis in this studyPrimer nameNucleotide sequences (5′–3′)PurposeOligo dT (OdT)GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT TcDNA synthesisOligo dT adapter (AP)GGC CAC GCG TCG ACT AGT ACHGAP-FwTGA AGG TGC TGT GTA TCC TCFragment PCRHGAP -RvCAG TAC TCT CGA TAG ACG ACHGAP -5′GAT TAC GCC AAG CTT ACA CGT CGA ACT TGC AGA CAG CGC CA5′ RACE PCRHGAP -3′GAT TAC GCC AAG CTT CTA CGT GAG CTT CTG TGC CTG GGA TC3′ RACE PCRHGAP -qFGGA ACA ATC TTT CGG CTA CGqRT-PCRHGAP -qRCAG TAC TCT CGA TAG ACG ACGnRH-qFATC​GCT​GCC​AGA​ACG​GAG​TGGnRH-qRCCA​CTG​TTA​CTG​CTA​CTA​CTGHdh-β-Actin-FwGAT AGT GCG AGA CAT CAA GGHdh-β-Actin-RvGAG CTC GAA ACC TCT CAT TGCloning of HGAP mRNA Sequence in Pacific AbaloneCloning of Partial SequenceFor partial sequence cloning of the Hdh-HGAP gene, reverse transcription polymerase chain reaction (RT-PCR) was performed using cDNA from mantle tissue, a gene-specific primer set (forward and reverse), and GoTaq® DNA Polymerase (Promega, Madison, WI, USA). The primer set used in this study was designed based on the known HGAP nucleotide sequence from H. asinina (GenBank Accession No. JN032738). The RT-PCR reaction mixture was prepared in a total volume of 50 μL, including cDNA (1 μL), 20 pmol of forward (HGAP-Fw) and reverse (HGAP-Rv) primers (1 μL each), GoTaq reaction buffer (colorless) (10 μL), dNTP mix (1 μL), DNA polymerase (0.25 μL), and ultra-pure water (35.75 μL). The RT-PCR thermal cycling conditions consisted of an initial denaturation step at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 45 s, with a final extension at 72°C for 5 min. After the RT-PCR, the products were subjected to gel electrophoresis using a 1.2% agarose gel, and the bands were purified using the Wizard® SV Gel and PCR Clean-Up System kit (Promega, Madison, WI, USA). The purified DNA was then ligated into the pGEM®-T Easy Vector (Promega) and transformed into DH5α chemically competent E. coli cells (Enzynomics, South Korea). Positive clones were selected for plasmid DNA purification using the Hybrid-QTM Plasmid Rapidprep mini kit (GeneAll, Seoul, South Korea) and sequenced at Macrogen (Seoul, South Korea).Cloning of RACE (5′- and 3′-) SequenceTo obtain the full-length sequence of Hdh-HGAP, RACE-PCR (rapid amplification of cDNA ends polymerase chain reaction) was conducted using the SMARTer® RACE 5′/3′ kit (Takara Bio Inc., Japan). Gene-specific 5′- and 3′-RACE primers were designed based on the previously obtained partial sequence of Hdh-HGAP, including a 15-bp overlap (GATTACGCCAAGCTT) at the 5′ end. For the 5′- and 3′-RACE-PCRs, cDNA (3′- or 5′-RACE) (2.5 μL), sense (3′-RACE) or antisense (5′-RACE) RACE primers (1 μL), SeqAmp DNA polymerase (1 μL), universal primer mix (UPM) (5 μL), SeqAmp buffer (25 μL), and PCR-grade water (15.5 μL) were used. A total of 30 cycles of touchdown PCR were performed for both 3′-RACE and 5′-RACE. The thermal cycling conditions were set according to the instructions provided by the kit manufacturer. The PCR products were analyzed by gel electrophoresis, and purification was carried out using the NucleoSpin® Gel and PCR Clean-up kit (MACHEREY-NAGEL GmbH & Co. KG, Germany). The purified products were ligated into a linearized pRACE vector and transformed into Stellar-competent cells (E. coli HST08). Positive clones were selected, purified, and sequenced at Macrogen, as described earlier for the partial sequence. Finally, the 5′-RACE sequence, the initially cloned partial cDNA fragment, and the 3′-RACE sequence were merged and trimmed to obtain the full-length Hdh-HGAP sequence.Sequence Analysis of Hdh-HGAP NeuropeptideThe complete nucleotide and amino acid sequence of the cloned Hdh-HGAP protein were analyzed using several online tools. The ORFfinder tool is available at https://www.ncbi.nlm.nih.gov/orffinder/ was used to predict potential protein-encoding segments and open reading frames (ORFs) from the nucleotide sequence. The ProtParam tool at https://web.expasy.org/protparam/ was utilized to determine the molecular weight and isoelectric point (pI) of the Hdh-HGAP protein sequence. SignalP 6.0 (https://services.healthtech.dtu.dk/services/SignalP-6.0/) and NeuroPred (http://stagbeetle.animal.uiuc.edu/cgi-bin/neuropred.py) were employed to identify any signal peptide and potential cleavage sites in the amino acid sequence. The online protein structure prediction server C-I-TASSER (https://zhanggroup.org/C-I-TASSER/) was used to analyze and predict the three-dimensional structure and function of the Hdh-HGAP protein. The Motif scan tool at https://myhits.sib.swiss/cgi-bin/motif_scan was utilized to predict any motifs present in the Hdh-HGAP sequence. Multiple sequence alignment was performed using MEGA (version 11.0.13) to compare the amino acid sequence of Pacific abalone HGAP with other abalone species, after which the results were visualized using the Jalview software (version 2.11.1.7).Phylogenetic AnalysisThe amino acid sequence of Hdh-HGAP was aligned with other HGAP and related uncharacterized protein sequences using the ClustalW online tool. Based on this alignment, a phylogenetic tree was constructed and edited using the MEGA software (version 11.0.13) with the maximum likelihood algorithm.Structure of Hdh-HGAP NeuropeptideThe three-dimensional (3D) structure of the Hdh-HGAP protein was predicted using the previously described C-I-TASSER server for protein structure and functional prediction. The resulting 3D structure was visualized using the UCSF ChimeraX software (version 1.2.5).Quantitative Real-Time PCR AnalysisTo quantify the relative mRNA expression levels of Hdh-HGAP, quantitative real-time PCR (qRT-PCR) analysis was performed on various tissues of Pacific abalone. The expression levels of Hdh-HGAP were measured in different adult abalone organs, as well as during different embryonic and larval developmental stages.All qRT-PCR assays were conducted following the guidelines provided by the 2×qPCRBIO SyGreen Mix Lo-Rox kit (PCR Biosystems Ltd., London, UK) manual. Each qRT-PCR reaction mixture was prepared in a total volume of 20 μL, consisting of cDNA template (1 μL), 10 pmol of gene-specific forward and reverse primers (1 μL each), SyGreen Mix (10 μL), and double-distilled water (10 μL). Triplicate reactions were performed for both the target and reference genes in each tissue sample. The PCR amplification conditions included an initial preincubation step at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 20 s, and extension at 72°C for 30 s. The melting temperature was set as the default setting of the instrument. Fluorescence readings were recorded at the end of each cycle for quantification. Amplification and data analysis were performed using a LightCycler® 96 System (Roche, Germany).Relative gene expression was determined using the 2−ΔΔCT method, with the Pacific abalone β-actin gene serving as an internal reference. All primers used for qRT-PCR analysis are summarized in Table 1.Statistical AnalysisThe mRNA expression values were subjected to statistical analysis and presented as the mean ± standard error. Changes in relative mRNA expression were calculated using nonparametric ANOVA analysis with the GraphPad Prism software (version 9.3.1). Statistical significance was defined as a p value of <0.05. All graphs were generated using Microsoft Excel and the GraphPad Prism 9.3.1 software.ResultsHdh-HGAP Sequence AnalysisThe cDNA sequence encoding H. discus hannai HGAP was obtained from the mantle tissue of H. discus hannai and designated as Hdh-HGAP. The full-length sequence of the Hdh-HGAP cDNA (GenBank accession No. OM937903) was 552 bp in length and included a long poly-A tail (Fig. 1). The 5′- and 3′-untranslated regions (UTRs) of Hdh-HGAP were 51 bp and 210 bp long, respectively. The open reading frame (ORF) of the Hdh-HGAP cDNA sequence was 291 bp, encoding 96 deduced amino acids. A putative polyadenylation signal (AATAAA) was identified in the nucleotide sequence, located 14 bp upstream of the poly-A tail. SignalP analysis predicted a 15-bp signal peptide at the N-terminal region, with a cleavage site between Glu16 and Glu17, indicating that the peptide is likely targeted to the secretory pathway. However, the NeuroPred tool predicted a propeptide cleavage site at residues Arg 25.Fig. 1.Full-length nucleotide and deduced amino acid sequences of Hdh-HGAP (GenBank accession No. OM937904.1). The numbers on the right and left sides indicate the amino acid and nucleotide positions, respectively. The start and stop codons are highlighted in green bold font and black bold font with a red asterisk, respectively. The signal peptide is shown in teal color. The N-myristoylation site is depicted in purple. The black box indicates the protein kinase C phosphorylation site. The red circles indicate the conserved cysteine residues, and the predicted cleavage site is indicated by a violet box. The polyadenylation signal is underlined.In motif scan analysis, a single N-myristoylation site (GAAVCE) with the structure G-(EDRKHPFYW) × (2)-(STAGCN)-(P) and a protein kinase C (PKC) phosphorylation site (TAK) with the structure (ST) × (RK) were detected at amino acid positions 12–17 and 19–21, respectively. In the multiple sequence alignment, nine cysteine residues were conserved when aligning the HGAP sequences of H. discus hannai, H. asinina, H. madaka, H. rubra, and H. rufescens (Fig. 2). The first 16 residues of the sequence were identified as the signal peptide, indicating that this protein is secreted. Additionally, the N-terminal region of Hdh-HGAP exhibited higher conservation compared to the C-terminal region across both vertebrates and invertebrates.Fig. 2.Multiple sequence alignment of Hdh-HGAP deduced amino acid sequences from H. discus hannai (UTD53615.1), H. asinina (AEW67131.1), H. madaka (ALU63745.1), H. rubra (XP 046545493.1), H. rufescens (XP 046343540.1), H. rufescens (XP 046343539.1), H. rubra (XP 046545496.1), and H. rubra (XP 046545495.1). The black box represents the signal peptide, and the red boxes highlight the conserved cysteine residues.The Hdh-HGAP protein exhibits a cysteine-rich structure. The predicted three-dimensional structure of Hdh-HGAP revealed that it is predominantly composed of a polypeptide chain rather than helices or beta-strands. Within the structure, a phosphate ligand was observed, which interacts with specific amino acids such as glutamine, tyrosine, and proline (Fig. 3).Fig. 3.Three-dimensional structure of Hdh-HGAP indicating ligand and terminal points.The phylogenetic tree was constructed using the maximum likelihood method based on the amino acid sequences of HGAP from Pacific abalone and other abalone species. Since HGAP is specific to abalone, the tree focused on the relationships between the amino acid sequences of various abalone species. Despite sharing more than 75% similarity and identity in their amino acid sequences, the abalone species formed two distinct clusters. The Hdh-HGAP sequence formed a clade with H. rufescens (XP 046343540.1), H. rubra (XP 046545493.1), and H. rubra (XP 046545495.1) with a high bootstrap value, indicating a close evolutionary relationship (Fig. 4).Fig. 4.Phylogenetic trees were constructed using the neighbor-joining method after ClustalW alignment, based on the amino acid residues of HGAP (characterized and uncharacterized) in different species of abalone. The numbers displayed at the nodes indicate the bootstrap probability. The phylogenetic tree was constructed using the following protein sequences and their accession IDs: H. discus hannai (UTD53615.1), H. rufescens (XP 046343540.1), H. rubra (XP 046545493.1), H. rubra (XP 046545495.1), H. asinina (AEW67131.1), H. rubra (XP 046545496.1), H. madaka (ALU63745.1), and H. rufescens (XP 046343539.1).Homology analysis based on the amino acid sequence revealed that Hdh-HGAP exhibited the highest similarity (96.87%) and identity (95.83%) with the amino acid sequences of H. rufescens and H. rufescens, whereas the lowest similarity and identity were observed with H. rubra (81.25% and 75%, respectively) (Table 2). It is worth noting that HGAP has been characterized in H. madaka and H. asinina, whereas the sequences of H. rufescens and H. rubra represent uncharacterized proteins.Table 2.Protein percent similarity and identity of HGAP protein (characterized and uncharacterized) in different abalone speciesProtein similarity percentagesH. discus hannai, %H. rufescens, %H. asinina, %H. rubra, %H. rufescens, %H. madaka, %H. rubra, %H. rubra, %H. discus hannai100H. rufescens96.87100H. asinina84.3783.33100H. rubra81.2581.2586.45100H. rufescens85.4184.3789.5884.37100H. madaka85.4184.3792.782.2996.87100H. rubra87.587.587.584.3787.586.45100H. discus hannai82.2982.2983.3379.1682.2982.2991.66100Protein identity percentagesH. discus hannai, %H. rufescens, %H. asinina, %H. rubra, %H. rufescens, %H. madaka, %H. rubra, %H. rubra, %H. discus hannai100H. rufescens95.83100H. asinina7576.04100H. rubra7577.0883.33100H. rufescens80.280.284.3781.25100H. madaka80.280.284.3779.1696.87100H. rubra81.2582.2978.1279.1680.278.12100H. rubra80.281.2578.1276.0477.0876.0487.5100Tissue-Specific mRNA Expression of Hdh-HGAPAmong the various tissues of Pacific abalone, the expression of Hdh-HGAP mRNA was found to be most significant (p < 0.05) in the muscle tissue, followed by the heart, pleuropedal ganglion, and mantle tissue. The lowest expression levels were observed in the gill tissue (Fig. 5). These results were consistent with the findings of our semi-quantitative RT-PCR expression analysis.Fig. 5.Hdh-HGAP mRNA expression level in different tissues of Pacific abalone H. discus hannai. CG, cerebral ganglion; PPG, pleuropedal ganglion; DG, digestive gland; TES, testis; OVR, ovary; MUS, muscle; MNT, mantle; GIL, gill; HRT, heart; HMC, hemocyte.Hdh-HGAP Expression in Embryonic and Larval Development of Pacific AbalonesDuring the embryonic and larval development stages of Pacific abalone, dynamic expression of Hdh-HGAP mRNA was observed. The expression levels of Hdh-HGAP mRNA were significantly lower in the early cell division stages (fertilized egg, 2-cells, 4-cells, morula) compared to unfertilized eggs, as well as larval (trochophore and veliger) and juvenile stages. However, after the cell division stage, the expression of Hdh-HGAP mRNA showed a significant increase (p < 0.05) during the trochophore larvae stage, followed by the veliger larvae and juvenile stages of Pacific abalone (Fig. 6).Fig. 6.Expression of Hdh-HGAP mRNA throughout the embryonic and larval developmental stages of Pacific abalone. FE, fertilized eggs; 2-CL, 2-cell; 4-CL, 4-cell; MOR, morula; TRP, trochophore; VLG, veliger; and JUV, juveniles, bf, before fertilization; hpf, hour of post-fertilization; ns, non-significant; *significant; ***highly significant.Hdh-HGAP Expression in Pacific Abalones Exhibiting Different Growth PatternsThe Hdh-HGAP mRNA exhibited upregulated expression according to the growth pattern, ranging from stunted growth to rapid growth. Notably, a significantly higher expression level (p < 0.05) was observed in abalone with rapid growth (Fig. 7). Conversely, the stunted-growth abalone displayed the lowest expression level, which was not significantly different from that of the abalone with minimal and normal growth.Fig. 7.Expression of Hdh-HGAP in mantle and muscle tissue of Pacific abalones exhibiting different growth patterns. SG, stunted growth; MG, minimal growth; NG, normal growth; RG, rapid growth; ns, non-significant; *significant; ***highly significant.Expression of Hdh-SPARC at Different Gonadal Development StagesThe expression of Hdh-HGAP mRNA varied significantly across the different developmental stages investigated in this study. The expression level was nearly negligible in the spent stage, followed by a slight increase in the proliferative stage. However, the expression was significantly higher (p < 0.05) in the ripening stage. Afterward, there was a significant decrease in Hdh-HGAP expression in the degenerative stage (Fig. 8).Fig. 8.Hdh-HGAP mRNA expression level at different gonadal development stages in Pacific abalone. SS, spent stage; PS, proliferative stage; RS, ripening stage; DS, degenerative stage; ns, non-significant; *significant; ***highly significant.In vivo Effects of GnRH Peptide on the Gonad Development and Expression of HGAPThe administration of GnRH peptide enhanced the gonad development process compared to the control treatment in Pacific abalone. Significant expression changes were observed in the CG, TES, and OVR tissues of male and female Pacific abalone in a time-dependent manner. Although the nature of expression for both GnRH and HGAP was similar, differences in expression were observed depending on time, organs, and sex. In male Pacific abalone, the expression of both GnRH and HGAP showed increasing trends until 30 days post-peptide injection, after which it decreased. Significantly higher expression was observed at 30 days post-peptide injection, followed by a subsequent decrease in both CG and TES tissue (Fig. 9a, c). However, the expression of GnRH was higher in CG compared to TES, while HGAP showed higher levels in TES compared to OVR. In contrast, a significant increase in expression was found at 15 days post-peptide administration in female Pacific abalone (Fig. 9b, d), which gradually decreased in a time-dependent manner. At the end of the experiment, the expression was similar to the control. In female Pacific abalone, the expression of GnRH was higher in CG compared to OVR, while HGAP showed comparatively higher levels in OVR than CG.Fig. 9.Relative mRNA expression levels of GnRH and HGAP in the CG, TES and OVR tissue of H. discus hannai after GnRH peptide treatment. a Male CG. b Female CG. c TES. d OVR. ns, non-significant; *significant; ***highly significant.Hdh-HGAP Expression during Muscle Remodeling of Pacific AbaloneExpression analysis of muscle-injured Pacific abalone revealed a significant (p < 0.05) upregulation of Hdh-HGAP mRNA from the first day of injury to 6 days (Fig. 10). After the sixth day, the expression of Hdh-HGAP negligibly decreased at 12 days. Subsequently, there was a rapid reduction in its expression at 24 days. The expression levels in the control and remodeled abalone were not significantly different. However, throughout the remodeling period, the expression of Hdh-HGAP remained higher compared to the control and remodeled abalones.Fig. 10.Expression of Hdh-HGAP in muscle-injured Pacific abalone H. discus hannai. ns, non-significant; *significant; ***highly significant.Expression of Hdh-HGAP during Starvation of Pacific AbaloneSignificant differences were observed in the mRNA expression of Hdh-HGAP in Pacific abalone during nutritional stress (starvation). The expression level gradually decreased as the starvation period increased, with the lowest expression observed in the third week (Fig. 11). However, after refeeding, a significantly higher expression of Hdh-HGAP was observed compared to the starvation period and control condition.Fig. 11.Expression of Hdh-HGAP in muscle tissue of starved Pacific abalone H. discus hannai. 1 W, 1 week; 2 W, 2 weeks; 3 W, 3 weeks; RF, refeeding. ns, non-significant; *significant; ***highly significant.DiscussionThe present study successfully identified the complete sequence of the neuropeptide Hdh-HGAP in Pacific abalone. Hdh-HGAP is a cysteine-rich protein without any distinct domains or signature motifs, except for a signal peptide at its N-terminal region. The presence of a single PKC phosphorylation site in the amino acid sequence suggests its role in regulating cell proliferation and gene expression [23]. The N-myristoylation site in the Hdh-HGAP sequence may play a crucial role in signaling and response to environmental stress [24]. The predicted signal peptide and cleavage site of Hdh-HGAP align with the previously published Has-HGAP amino acid sequence, although there are some differences in the amino acid residues [18]. The phosphate (PO4) ligand within the protein structure is important for phosphorylation and dephosphorylation processes in living organisms [25]. Amino acid sequence analysis revealed that Hdh-HGAP shares more than 80% similarity with previously identified HGAP sequences from H. asinina (Has-HGAP) and H. madaka (Hma-HGAP). Due to the species-specific (abalone) nature of HGAP, comparison with other species was not possible during phylogenetic analysis. However, among the available amino acid sequences in Haliotis species, Hdh-HGAP clustered with the uncharacterized HGAP sequence of H. rufescens and two sequences of H. rubra. The other cluster consisted of two characterized sequences (H. asinina and H. madaka) and two uncharacterized sequences (H. rufescens and H. rubra). The difference between these clusters is based on the percentage of protein identity, whereas the number of amino acid residues (96) remains constant for this neuropeptide.Despite being a neuropeptide, Hdh-HGAP exhibited significant expression in muscle tissue during tissue-specific expression analysis. It is important to note that the activity of a gene is not limited to the tissue in which it is secreted, and the expression of a gene in the endocrine system can have phenotypic consequences in other tissues due to its secretory function [26]. A previous study on the growth regulatory neuropeptide of H. asinina Has-HGAP also reported similar expression patterns, with higher expression in muscle and mantle compared to secretory organs such as cerebral and pleuropedal ganglia and no expression in gills [18]. Consistent with these findings, the present study found the lowest expression of Hdh-HGAP in gill tissue. Although Hdh-HGAP is known to be involved in growth and reproductive functions, its expression in the gonad (TES and OVR) was comparatively low. It is worth mentioning that the tissue samples for tissue-specific expression analysis were collected during the spent/resting stage of gonadal development, which could explain the lower expression in the gonad. A gene is expressed when it is needed in the cells to take part in a certain function or mechanism [27].During early embryonic and larval development, Hdh-HGAP exhibited minimal expression during cell division stages (2-cell, 4-cell, morula), while higher expression was observed in trochophore, veliger, and juvenile stages compared to the control (unfertilized egg). As this is the first report of Hdh-HGAP expression during the metamorphosis of Pacific abalone, the exact reasons for these expression patterns are not fully understood. However, it is known that significant morphological and behavioral changes occur during the trochophore and veliger stages, including the formation of cilia, free swimming, retractor muscle development, mantle and foot mass formation, and shell development [28]. Therefore, it is plausible that Hdh-HGAP may have direct or indirect involvement in these developmental events. In fact, previous studies have reported the involvement of secreted growth-related genes in the metamorphosis of Pacific abalone [22].In the present study, rapidly growing juvenile Pacific abalone exhibited higher Hdh-HGAP expression compared to their stunted growth, minimum growth, and normal growth counterparts. Similar observations were made in H. asinina, where Has-HGAP was extensively expressed in fast-growing abalone compared to slow-growing abalone [18]. By comparing these findings with the known role of ganglionic neuropeptides in growth regulation in other gastropods [29‒31], we concluded that the expression of Hdh-HGAP reflects the physiological state of Pacific abalone. However, the underlying mechanism is currently unknown.In general, reproductive neuropeptides are remarkably expressed during reproductive season. The expression can vary based on sex during cell functioning [32]. The higher expression in the TES compared to the OVR suggests that Hdh-HGAP may play a more prominent role in regulating male reproductive activity than in females in Pacific abalone. Similar study reported that Has-HGAP controls the reproductive activity of male and female H. asinina, where male expression was comparatively higher [11]. The expression level of Hdh-HGAP was relatively lower compared to other reproductive regulatory neuropeptides such as Has-APGWamide, Has-Myomodulin, and Has-Whitnin. Nonetheless, a recent transcriptome and peptidome analysis in neural ganglia related to sexual maturation did not detect HGAP in Pacific abalone [10].Peptide administration influences gonad development and enhances the expression of both GnRH and HGAP in male and female Pacific abalones. In a previous study, Rajib et al. [32] reported that GnRH is responsible for the gonadal cell proliferation of both male and female Pacific abalone. Typically, hormones responsible for regulating a particular function exhibit analogous expression patterns, influenced by developmental stages, timing, and dosages (when external substances are administered) [32]. The current study reports that HGAP expression exhibits a pattern similar to GnRH in a time-dependent manner when synthetic GnRH peptide is injected. This phenomenon indicates that HGAP possibly participates in the gonad development of Pacific abalone as like other reproductive neuropeptide. While in H. asinina, there was a negligible difference in HGAP expression between the male and female anterior ganglion (CG) [11], in the case of Pacific abalone, alterations in the expression of Hdh-HGAP are noticeable depending on the sex and organs.During muscle tissue remodeling, qRT-PCR expression analysis showed that Hdh-HGAP was highly expressed throughout the repair process, with reduced expression observed at the end of remodeling. These findings suggest that the Hdh-HGAP neuropeptide may be involved in tissue remodeling following damage. Although the involvement of neuropeptides in tissue remodeling is not well studied, previous research has shown that certain neuropeptides directly or indirectly regulate tissue repair [33]. For example, Substance-P has been found to directly induce tissue repair by activating the PI3K/Akt/mTOR pathway even in the presence of IFNγ [34]. However, the receptor for HGAP and its regulatory pathway have not yet been identified, and therefore the specific mechanisms through which Hdh-HGAP participates in tissue remodeling in Pacific abalone remain unknown.Starvation was found to affect the activity of the Hdh-HGAP neuropeptide gene. During the early stages of starvation, the expression of Hdh-HGAP gradually decreased with increasing starvation duration, and a significant increase in expression was observed after refeeding. These observations suggest that starvation may negatively regulate the expression of Hdh-HGAP in Pacific abalone. During starvation, abalone are deprived of proper food and nutrients, which can potentially impact the expression of Hdh-HGAP. Although the effects of starvation on neuropeptide activity have been extensively studied [35‒37], our study is the first to report on the effects of starvation on Hdh-HGAP neuropeptide expression. Previous research found upregulated expression of HGAP in the neural ganglia of well-nourished H. asinina individuals compared to unfed individuals [18].ConclusionThe present study provides important insights into the identification and characterization of the HGAP neuropeptide in Pacific abalone, revealing its significant expression differences in various physiological processes. The inheritance of growth rate and the known role of neurosecretory cells in controlling growth in other mollusks suggest a potential involvement of Hdh-HGAP in promoting growth. The differential expression of Hdh-HGAP in different reproductive stages and peptide administration implies its potential role in regulating reproduction. The observed differential expression during metamorphosis highlights its regulatory role in this critical developmental process. Moreover, the upregulation of mRNA expression during muscle tissue remodeling and its downregulation during starvation suggest that Hdh-HGAP may play a role in tissue repair and that its expression is negatively regulated by starvation. Collectively, these findings suggest that Hdh-HGAP plays a multifaceted role in regulating growth, reproduction, metamorphosis, tissue damage repair, and the response to starvation in Pacific abalone.Statement of EthicsThe experimental protocols used in this study were approved by the Animal Care and Use Committee of Chonnam National University (approval number: CNU IACUC-YS-2020-5). The present study was conducted following the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health.Conflict of Interest StatementThe authors have no conflicts of interest to disclose.Funding SourcesThis research was supported by a grant (20180375) funded by the Ministry of Oceans and Fisheries, South Korea.Author ContributionsConceptualization, investigation, and writing-original draft preparation: K.H.K. and M.A.H.; methodology: M.A.H., S.H., D.H.C., and K.H.K.; formal analysis: M.A.H.; visualization and data curation: M.A.H. and S.H.; and validation, resources, writing-review and editing, project administration, funding acquisition, and supervision: K.H.K. All authors read and agreed to the published version of the manuscript. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Neuroendocrinology Karger

The Neuropeptide HGAP Regulates Growth, Reproduction, Metamorphosis, Tissue Damage Repair, and Response against Starvation in Pacific Abalone

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Karger
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© 2023 The Author(s). Published by S. Karger AG, Basel
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0028-3835
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1423-0194
DOI
10.1159/000535945
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Abstract

IntroductionNeuroendocrine systems play vital roles in regulating growth and reproduction by releasing amino acid derivatives, proteins, and peptides [1]. In mollusks, the neural ganglia secrete numerous neuropeptides that regulate brain activity in response to signals. The cerebral and pleuropedal ganglia are particularly important for producing neuropeptides involved in mollusk growth and reproduction [2, 3]. However, the activity of a neuropeptide may not be limited to growth and reproduction; it may exhibit diverse physiological functions in the organism.Growth of all living organisms is influenced by genetic, nutritional, and environmental factors [4]. In vertebrates, growth is primarily regulated by the growth hormone (GH) and insulin-like factor (IGF) axes, which are secreted by neuroendocrine cells [5]. Although GH has not been identified in invertebrates, insulin-like peptides and related derivatives are known to play key roles in various growth-related processes, including glucose, lipid, and amino acid metabolism in mollusks [6, 7]. In addition to GH and IGFs, species-specific growth factors and peptides contribute to growth regulation in different organisms.Neuroendocrine components control cell signal communication and play crucial roles in gamete formation, regulation, maturation, and release in both vertebrates and invertebrates [8]. Neural ganglia produce neuropeptides that act as reproductive regulators and participate in the reproductive process [9, 10]. The regulation of the reproductive cycle is attributed, at least in part, to a complex combination of neurohormones rather than a single peptide [11]. Detailed analyses of reproduction-related genes and their functions are being carried out in various mollusk species, such as freshwater snails, cuttlefish, and oysters, using transcriptomic resources [12‒16]. Gonadotropin-releasing hormone (GnRH) is a major neuroendocrine hormone that plays a central role in controlling reproductive process in abalone. While GnRH is a representative neuropeptide for regulating reproductive function in vertebrates and invertebrates, other neuropeptides such as caudo-dorsal cell hormone, FMRFamide, and APGWamide also play roles in various reproductive activities [17].In addition to these neuropeptides, there are some species-specific neuropeptides, such as molluscan insulin-related peptide (MIP) and haliotid growth-associated peptide (HGAP), which have been reported to regulate both growth and reproduction in mollusks [11, 18]. Among these, HGAP is specific to abalone. Previous research first identified the HGAP neuropeptide in Haliotis asinina, where it was found to be upregulated in the neural ganglia. In the context of abalone production, there is considerable interest in improving growth rates and controlling reproduction.Despite losses in abalone capture yields in natural environments, abalone aquaculture has experienced significant growth worldwide in recent decades [19, 20]. In South Korea, abalone aquaculture production reached 23,199 tons in 2021, accounting for 30% of the country’s shellfish production [21]. As abalone aquaculture relies entirely on hatchery-produced seed, understanding the reproductive behavior and regulation of abalone is crucial for successful seed production. One of the major challenges in abalone aquaculture, both in farm- and sea cage-based cultures, is achieving desirable growth rates. The growth and reproduction of abalone are genetically regulated by various hormones, peptides, and their derivatives related to growth and reproduction. While most of these molecules have specific functions in either growth or reproduction regulation, some may exhibit multifunctional roles. A recent transcriptome study on the ass’s-ear abalone H. asinina identified a specific neuropeptide called HGAP, which is reported to regulate both growth and reproduction of abalone. However, there have been no studies characterizing this neuropeptide and exploring its diverse regulatory roles in Pacific abalone. Considering that Pacific abalone (Haliotis discus hannai) is the most commercially cultured marine gastropod species in South Korea, this study aimed to clone, characterize, and investigate the regulatory role of HGAP in Pacific abalone. Particularly, this study will focus on various aspects such as growth, metamorphosis, muscle tissue remodeling, response to starvation, and reproduction, using expression analysis.MethodsExperimental AnimalsThree-year-old sexually mature Pacific abalones with an average body weight of 120.4 ± 0.61 g and an average shell length of 84.06 ± 0.32 mm were collected from sea cages in Jindo-gun, South Korea. After carefully observing the health status of abalones in different age groups, we have identified this three-year-old Pacific abalone as suitable for the present study. The abalones were transported using oxygenated plastic tanks to the Tou-Jeong Soosan abalone hatchery in Dolsan-eup, Yeosu, South Korea, where they were cultured for 1 month with sufficient food and water supply to recover from the transportation-related stress. After adjustment, these abalones were used for cloning and tissue-specific expression analysis, as well as for artificial breeding, GnRH peptide treatment, starvation treatment, and experiments related to muscle tissue remodeling.Tissue Collection for Gene Cloning and Expression AnalysisFor cloning and tissue-specific expression analysis, a total of 12 Pacific abalones were sacrificed, and their hemocytes (HMC), cerebral ganglion (CG), pleuropedal ganglion (PPG), testis (TES), ovary (OVR), mantle (MNT), muscle (MUS), gill (GIL), heart (HRT), and digestive gland (DG) tissues were collected. Prior to tissue sample collection, the abalones were anesthetized using 5% MgCl2. The collected tissue samples were washed with phosphate-buffered saline (PBS; 0.1 m), immediately snap-frozen in liquid nitrogen (LN2), and stored at −80°C until total RNA extraction.Embryo and Larvae CollectionThree-year-old reproductively mature 12 females’ and 4 male’s abalone were induced for artificial fertilization. After spawning, unfertilized eggs (UFE) and sperm were collected, and artificial fertilization was performed following the procedure described by Hanif et al. [22]. After fertilization, samples of target stages, including fertilized eggs (FE), 2-cell (2-CL) and 4-cell (4-CL) stages, morula (MOR), trochophore (TRP) larvae, veliger (VLG) larvae, and juveniles (JUV), were collected through microscopic observation. The juvenile sample was collected monthly for up to 6 months. The samples were immediately snap-frozen in LN2 and stored at −80°C until total RNA extraction.Juvenile Abalone Culture and Growth Type Sample CollectionAfter fertilization, settlement larvae were transferred to a culture tank with diatoms precoated on a transparent and flat plastic film. For successful settlement of larvae in the tank and plastic sheet, we provided enough aeration in the tank, but seawater supply was stopped on the first day of larvae release to avoid washout of larvae. The early juvenile abalones were fed diatoms on plastic films and provided artificial powder feed (composed of kelp powder, fish meal powder, micronutrients, and effective microorganisms) for 2 months. In South Korea, several artificial powder feeds and granular feeds of different sizes are available for abalone nursing at the farm level. Importantly, artificial feed has been shown to perform better than diets exclusively based on algae in abalone aquaculture. After 2 months, we switched from powder feed to granular feed (2 × 1 mm) for an additional 2 months. Next, 3 × 1 mm granular feed was provided for 1 year. After 6 months, juvenile abalones were collected and sorted according to size. Afterward, length, width, and weight data were taken, tagged, and again reared in the tank for 1 year. One year later, we collected all the abalones, measured them, and compared them with previous data. After comparison, we categorized them as follows: SG, stunted growth (size increased >3 mm); MG, minimal growth (size increased 5 mm to >1 cm); NG, normal growth (size increased 1–2 cm); and RG, rapid growth (size increased <2 cm). Then, muscle tissue samples of five juveniles from each category were collected. The samples were flash-frozen in LN2 and stored at −80°C until total RNA extraction.Tissue Sample from Injured Pacific AbaloneThree-year-old mature Pacific abalones (n = 40) with an average body weight of 121.2 ± 0.53 g and an average shell length of 84.11 ± 0.51 mm were used for the muscle tissue remodeling experiment. The abalones were divided into two groups where group 1 served as control and group 2 as for remodeling treatment. Briefly, 3 × 1 mm pieces of muscle tissue were carefully removed from 25 abalones using a tissue-collecting needle after anesthetizing them with 5% MgCl2. Subsequently, they were cultured in a rearing tank with continuous aeration, water, and a food supply. Muscle tissue samples from the injured region of three abalones were collected at four time points: 1 day, 6 days, 12 days, and 24 days. Additionally, tissue samples from control (initial) abalones and remodeled abalones (after 42 days of injury) were collected. All samples were washed with PBS, immediately snap-frozen in LN2, and stored at −80°C until total RNA extraction.Tissue Collection at Different Gonad Developmental StagesIn the reproductive season, samples of CG and gonad tissue (TES and OVR) were collected at different gonadal development stages, including the spent stage (SS), proliferative stage (PS), ripening stage (RS), and degenerative stage (DS). The tissue samples were immediately flash-frozen in LN2 and stored at −80°C until total RNA extraction.Administration of GnRH Peptide in the Gonad of Pacific AbaloneDuring the resting/spent stage (when gonad was completely empty), adult male (n = 20, body weight 123.43 ± 4.6 g, shell length 84.24 ± 2.6 mm) and female (n = 20, body weight 121.52 ± 4.9 g, shell length 83.94 ± 2.6 mm) were reared in the cemented tank with running sea water in the hatchery. Forty individuals were divided into four groups (two female groups and two male groups). Group 1 (male) and 3 (female) were considered control, treated with PBS. Group 2 (male) and 4 (female) were injected with a synthetic GnRH peptide (pQNYHFSNGWYA-NH2) of Pacific abalone (accession No. AZL93822.1), generated by ANYGEN (Gwangju, South Korea) at dose of 250 ng/g body weight into the pedal sinus of Pacific abalone. The water temperature was maintained at 22 ± 0.6°C during the experiment. The CG, TES, and OVR samples from each group were randomly collected from five individuals after 15 days, 30 days, and 45 days. All samples were washed with PBS, immediately snap-frozen in LN2, and stored at −80°C until total RNA extraction.Tissue Samples from Starved Pacific AbalonesThree-year-old Pacific abalone (n = 30, shell length 84.43 ± 0.62 mm; body mass 121.7 ± 3.36 g) were randomly collected from the sea cages in Wando-gun, South Korea, and transported to the abalone hatchery in Yeosu, South Korea. The collected abalones were reared in tanks with running seawater flow, aeration, and adequate feed supply for adjustment. The abalones were divided into two groups, where group A served as control and group B for starvation treatment. At the beginning of starvation treatment, muscle tissue from three control individuals was collected. Since then, group B abalones were reared without feeding, and muscle tissue was collected from three abalones at 1-week intervals for 3 weeks. Afterward, starved abalones were reared with feed for 1 day, and samples were collected. Samples from control abalones were also collected at each sampling period following the procedure mentioned above and stored at −80°C until total RNA extraction.Total RNA Extraction and First-Stand cDNA SynthesisTotal cellular RNA was extracted from the collected tissue samples using the ISOSPIN Cell and Tissue RNA Kit (Nippon Gene, Tokyo, Japan). First-strand cDNA synthesis was performed using extracted RNA, oligo(dt) primers (Sigma), and the Superscript III First-Strand cDNA Synthesis Kit (Invitrogen, USA) (Table 1). The RACE cDNA (3′- and 5′-RACE) was synthesized from the extracted total RNA using the SMARTer® RACE 5′/3′ Kit (Takara Bio Inc., Japan). Both total RNA extraction and cDNA synthesis were conducted according to the manufacturer’s instructions.Table 1.List of different primers used for cDNA synthesis, cloning, and expression analysis in this studyPrimer nameNucleotide sequences (5′–3′)PurposeOligo dT (OdT)GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT TcDNA synthesisOligo dT adapter (AP)GGC CAC GCG TCG ACT AGT ACHGAP-FwTGA AGG TGC TGT GTA TCC TCFragment PCRHGAP -RvCAG TAC TCT CGA TAG ACG ACHGAP -5′GAT TAC GCC AAG CTT ACA CGT CGA ACT TGC AGA CAG CGC CA5′ RACE PCRHGAP -3′GAT TAC GCC AAG CTT CTA CGT GAG CTT CTG TGC CTG GGA TC3′ RACE PCRHGAP -qFGGA ACA ATC TTT CGG CTA CGqRT-PCRHGAP -qRCAG TAC TCT CGA TAG ACG ACGnRH-qFATC​GCT​GCC​AGA​ACG​GAG​TGGnRH-qRCCA​CTG​TTA​CTG​CTA​CTA​CTGHdh-β-Actin-FwGAT AGT GCG AGA CAT CAA GGHdh-β-Actin-RvGAG CTC GAA ACC TCT CAT TGCloning of HGAP mRNA Sequence in Pacific AbaloneCloning of Partial SequenceFor partial sequence cloning of the Hdh-HGAP gene, reverse transcription polymerase chain reaction (RT-PCR) was performed using cDNA from mantle tissue, a gene-specific primer set (forward and reverse), and GoTaq® DNA Polymerase (Promega, Madison, WI, USA). The primer set used in this study was designed based on the known HGAP nucleotide sequence from H. asinina (GenBank Accession No. JN032738). The RT-PCR reaction mixture was prepared in a total volume of 50 μL, including cDNA (1 μL), 20 pmol of forward (HGAP-Fw) and reverse (HGAP-Rv) primers (1 μL each), GoTaq reaction buffer (colorless) (10 μL), dNTP mix (1 μL), DNA polymerase (0.25 μL), and ultra-pure water (35.75 μL). The RT-PCR thermal cycling conditions consisted of an initial denaturation step at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 45 s, with a final extension at 72°C for 5 min. After the RT-PCR, the products were subjected to gel electrophoresis using a 1.2% agarose gel, and the bands were purified using the Wizard® SV Gel and PCR Clean-Up System kit (Promega, Madison, WI, USA). The purified DNA was then ligated into the pGEM®-T Easy Vector (Promega) and transformed into DH5α chemically competent E. coli cells (Enzynomics, South Korea). Positive clones were selected for plasmid DNA purification using the Hybrid-QTM Plasmid Rapidprep mini kit (GeneAll, Seoul, South Korea) and sequenced at Macrogen (Seoul, South Korea).Cloning of RACE (5′- and 3′-) SequenceTo obtain the full-length sequence of Hdh-HGAP, RACE-PCR (rapid amplification of cDNA ends polymerase chain reaction) was conducted using the SMARTer® RACE 5′/3′ kit (Takara Bio Inc., Japan). Gene-specific 5′- and 3′-RACE primers were designed based on the previously obtained partial sequence of Hdh-HGAP, including a 15-bp overlap (GATTACGCCAAGCTT) at the 5′ end. For the 5′- and 3′-RACE-PCRs, cDNA (3′- or 5′-RACE) (2.5 μL), sense (3′-RACE) or antisense (5′-RACE) RACE primers (1 μL), SeqAmp DNA polymerase (1 μL), universal primer mix (UPM) (5 μL), SeqAmp buffer (25 μL), and PCR-grade water (15.5 μL) were used. A total of 30 cycles of touchdown PCR were performed for both 3′-RACE and 5′-RACE. The thermal cycling conditions were set according to the instructions provided by the kit manufacturer. The PCR products were analyzed by gel electrophoresis, and purification was carried out using the NucleoSpin® Gel and PCR Clean-up kit (MACHEREY-NAGEL GmbH & Co. KG, Germany). The purified products were ligated into a linearized pRACE vector and transformed into Stellar-competent cells (E. coli HST08). Positive clones were selected, purified, and sequenced at Macrogen, as described earlier for the partial sequence. Finally, the 5′-RACE sequence, the initially cloned partial cDNA fragment, and the 3′-RACE sequence were merged and trimmed to obtain the full-length Hdh-HGAP sequence.Sequence Analysis of Hdh-HGAP NeuropeptideThe complete nucleotide and amino acid sequence of the cloned Hdh-HGAP protein were analyzed using several online tools. The ORFfinder tool is available at https://www.ncbi.nlm.nih.gov/orffinder/ was used to predict potential protein-encoding segments and open reading frames (ORFs) from the nucleotide sequence. The ProtParam tool at https://web.expasy.org/protparam/ was utilized to determine the molecular weight and isoelectric point (pI) of the Hdh-HGAP protein sequence. SignalP 6.0 (https://services.healthtech.dtu.dk/services/SignalP-6.0/) and NeuroPred (http://stagbeetle.animal.uiuc.edu/cgi-bin/neuropred.py) were employed to identify any signal peptide and potential cleavage sites in the amino acid sequence. The online protein structure prediction server C-I-TASSER (https://zhanggroup.org/C-I-TASSER/) was used to analyze and predict the three-dimensional structure and function of the Hdh-HGAP protein. The Motif scan tool at https://myhits.sib.swiss/cgi-bin/motif_scan was utilized to predict any motifs present in the Hdh-HGAP sequence. Multiple sequence alignment was performed using MEGA (version 11.0.13) to compare the amino acid sequence of Pacific abalone HGAP with other abalone species, after which the results were visualized using the Jalview software (version 2.11.1.7).Phylogenetic AnalysisThe amino acid sequence of Hdh-HGAP was aligned with other HGAP and related uncharacterized protein sequences using the ClustalW online tool. Based on this alignment, a phylogenetic tree was constructed and edited using the MEGA software (version 11.0.13) with the maximum likelihood algorithm.Structure of Hdh-HGAP NeuropeptideThe three-dimensional (3D) structure of the Hdh-HGAP protein was predicted using the previously described C-I-TASSER server for protein structure and functional prediction. The resulting 3D structure was visualized using the UCSF ChimeraX software (version 1.2.5).Quantitative Real-Time PCR AnalysisTo quantify the relative mRNA expression levels of Hdh-HGAP, quantitative real-time PCR (qRT-PCR) analysis was performed on various tissues of Pacific abalone. The expression levels of Hdh-HGAP were measured in different adult abalone organs, as well as during different embryonic and larval developmental stages.All qRT-PCR assays were conducted following the guidelines provided by the 2×qPCRBIO SyGreen Mix Lo-Rox kit (PCR Biosystems Ltd., London, UK) manual. Each qRT-PCR reaction mixture was prepared in a total volume of 20 μL, consisting of cDNA template (1 μL), 10 pmol of gene-specific forward and reverse primers (1 μL each), SyGreen Mix (10 μL), and double-distilled water (10 μL). Triplicate reactions were performed for both the target and reference genes in each tissue sample. The PCR amplification conditions included an initial preincubation step at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 20 s, and extension at 72°C for 30 s. The melting temperature was set as the default setting of the instrument. Fluorescence readings were recorded at the end of each cycle for quantification. Amplification and data analysis were performed using a LightCycler® 96 System (Roche, Germany).Relative gene expression was determined using the 2−ΔΔCT method, with the Pacific abalone β-actin gene serving as an internal reference. All primers used for qRT-PCR analysis are summarized in Table 1.Statistical AnalysisThe mRNA expression values were subjected to statistical analysis and presented as the mean ± standard error. Changes in relative mRNA expression were calculated using nonparametric ANOVA analysis with the GraphPad Prism software (version 9.3.1). Statistical significance was defined as a p value of <0.05. All graphs were generated using Microsoft Excel and the GraphPad Prism 9.3.1 software.ResultsHdh-HGAP Sequence AnalysisThe cDNA sequence encoding H. discus hannai HGAP was obtained from the mantle tissue of H. discus hannai and designated as Hdh-HGAP. The full-length sequence of the Hdh-HGAP cDNA (GenBank accession No. OM937903) was 552 bp in length and included a long poly-A tail (Fig. 1). The 5′- and 3′-untranslated regions (UTRs) of Hdh-HGAP were 51 bp and 210 bp long, respectively. The open reading frame (ORF) of the Hdh-HGAP cDNA sequence was 291 bp, encoding 96 deduced amino acids. A putative polyadenylation signal (AATAAA) was identified in the nucleotide sequence, located 14 bp upstream of the poly-A tail. SignalP analysis predicted a 15-bp signal peptide at the N-terminal region, with a cleavage site between Glu16 and Glu17, indicating that the peptide is likely targeted to the secretory pathway. However, the NeuroPred tool predicted a propeptide cleavage site at residues Arg 25.Fig. 1.Full-length nucleotide and deduced amino acid sequences of Hdh-HGAP (GenBank accession No. OM937904.1). The numbers on the right and left sides indicate the amino acid and nucleotide positions, respectively. The start and stop codons are highlighted in green bold font and black bold font with a red asterisk, respectively. The signal peptide is shown in teal color. The N-myristoylation site is depicted in purple. The black box indicates the protein kinase C phosphorylation site. The red circles indicate the conserved cysteine residues, and the predicted cleavage site is indicated by a violet box. The polyadenylation signal is underlined.In motif scan analysis, a single N-myristoylation site (GAAVCE) with the structure G-(EDRKHPFYW) × (2)-(STAGCN)-(P) and a protein kinase C (PKC) phosphorylation site (TAK) with the structure (ST) × (RK) were detected at amino acid positions 12–17 and 19–21, respectively. In the multiple sequence alignment, nine cysteine residues were conserved when aligning the HGAP sequences of H. discus hannai, H. asinina, H. madaka, H. rubra, and H. rufescens (Fig. 2). The first 16 residues of the sequence were identified as the signal peptide, indicating that this protein is secreted. Additionally, the N-terminal region of Hdh-HGAP exhibited higher conservation compared to the C-terminal region across both vertebrates and invertebrates.Fig. 2.Multiple sequence alignment of Hdh-HGAP deduced amino acid sequences from H. discus hannai (UTD53615.1), H. asinina (AEW67131.1), H. madaka (ALU63745.1), H. rubra (XP 046545493.1), H. rufescens (XP 046343540.1), H. rufescens (XP 046343539.1), H. rubra (XP 046545496.1), and H. rubra (XP 046545495.1). The black box represents the signal peptide, and the red boxes highlight the conserved cysteine residues.The Hdh-HGAP protein exhibits a cysteine-rich structure. The predicted three-dimensional structure of Hdh-HGAP revealed that it is predominantly composed of a polypeptide chain rather than helices or beta-strands. Within the structure, a phosphate ligand was observed, which interacts with specific amino acids such as glutamine, tyrosine, and proline (Fig. 3).Fig. 3.Three-dimensional structure of Hdh-HGAP indicating ligand and terminal points.The phylogenetic tree was constructed using the maximum likelihood method based on the amino acid sequences of HGAP from Pacific abalone and other abalone species. Since HGAP is specific to abalone, the tree focused on the relationships between the amino acid sequences of various abalone species. Despite sharing more than 75% similarity and identity in their amino acid sequences, the abalone species formed two distinct clusters. The Hdh-HGAP sequence formed a clade with H. rufescens (XP 046343540.1), H. rubra (XP 046545493.1), and H. rubra (XP 046545495.1) with a high bootstrap value, indicating a close evolutionary relationship (Fig. 4).Fig. 4.Phylogenetic trees were constructed using the neighbor-joining method after ClustalW alignment, based on the amino acid residues of HGAP (characterized and uncharacterized) in different species of abalone. The numbers displayed at the nodes indicate the bootstrap probability. The phylogenetic tree was constructed using the following protein sequences and their accession IDs: H. discus hannai (UTD53615.1), H. rufescens (XP 046343540.1), H. rubra (XP 046545493.1), H. rubra (XP 046545495.1), H. asinina (AEW67131.1), H. rubra (XP 046545496.1), H. madaka (ALU63745.1), and H. rufescens (XP 046343539.1).Homology analysis based on the amino acid sequence revealed that Hdh-HGAP exhibited the highest similarity (96.87%) and identity (95.83%) with the amino acid sequences of H. rufescens and H. rufescens, whereas the lowest similarity and identity were observed with H. rubra (81.25% and 75%, respectively) (Table 2). It is worth noting that HGAP has been characterized in H. madaka and H. asinina, whereas the sequences of H. rufescens and H. rubra represent uncharacterized proteins.Table 2.Protein percent similarity and identity of HGAP protein (characterized and uncharacterized) in different abalone speciesProtein similarity percentagesH. discus hannai, %H. rufescens, %H. asinina, %H. rubra, %H. rufescens, %H. madaka, %H. rubra, %H. rubra, %H. discus hannai100H. rufescens96.87100H. asinina84.3783.33100H. rubra81.2581.2586.45100H. rufescens85.4184.3789.5884.37100H. madaka85.4184.3792.782.2996.87100H. rubra87.587.587.584.3787.586.45100H. discus hannai82.2982.2983.3379.1682.2982.2991.66100Protein identity percentagesH. discus hannai, %H. rufescens, %H. asinina, %H. rubra, %H. rufescens, %H. madaka, %H. rubra, %H. rubra, %H. discus hannai100H. rufescens95.83100H. asinina7576.04100H. rubra7577.0883.33100H. rufescens80.280.284.3781.25100H. madaka80.280.284.3779.1696.87100H. rubra81.2582.2978.1279.1680.278.12100H. rubra80.281.2578.1276.0477.0876.0487.5100Tissue-Specific mRNA Expression of Hdh-HGAPAmong the various tissues of Pacific abalone, the expression of Hdh-HGAP mRNA was found to be most significant (p < 0.05) in the muscle tissue, followed by the heart, pleuropedal ganglion, and mantle tissue. The lowest expression levels were observed in the gill tissue (Fig. 5). These results were consistent with the findings of our semi-quantitative RT-PCR expression analysis.Fig. 5.Hdh-HGAP mRNA expression level in different tissues of Pacific abalone H. discus hannai. CG, cerebral ganglion; PPG, pleuropedal ganglion; DG, digestive gland; TES, testis; OVR, ovary; MUS, muscle; MNT, mantle; GIL, gill; HRT, heart; HMC, hemocyte.Hdh-HGAP Expression in Embryonic and Larval Development of Pacific AbalonesDuring the embryonic and larval development stages of Pacific abalone, dynamic expression of Hdh-HGAP mRNA was observed. The expression levels of Hdh-HGAP mRNA were significantly lower in the early cell division stages (fertilized egg, 2-cells, 4-cells, morula) compared to unfertilized eggs, as well as larval (trochophore and veliger) and juvenile stages. However, after the cell division stage, the expression of Hdh-HGAP mRNA showed a significant increase (p < 0.05) during the trochophore larvae stage, followed by the veliger larvae and juvenile stages of Pacific abalone (Fig. 6).Fig. 6.Expression of Hdh-HGAP mRNA throughout the embryonic and larval developmental stages of Pacific abalone. FE, fertilized eggs; 2-CL, 2-cell; 4-CL, 4-cell; MOR, morula; TRP, trochophore; VLG, veliger; and JUV, juveniles, bf, before fertilization; hpf, hour of post-fertilization; ns, non-significant; *significant; ***highly significant.Hdh-HGAP Expression in Pacific Abalones Exhibiting Different Growth PatternsThe Hdh-HGAP mRNA exhibited upregulated expression according to the growth pattern, ranging from stunted growth to rapid growth. Notably, a significantly higher expression level (p < 0.05) was observed in abalone with rapid growth (Fig. 7). Conversely, the stunted-growth abalone displayed the lowest expression level, which was not significantly different from that of the abalone with minimal and normal growth.Fig. 7.Expression of Hdh-HGAP in mantle and muscle tissue of Pacific abalones exhibiting different growth patterns. SG, stunted growth; MG, minimal growth; NG, normal growth; RG, rapid growth; ns, non-significant; *significant; ***highly significant.Expression of Hdh-SPARC at Different Gonadal Development StagesThe expression of Hdh-HGAP mRNA varied significantly across the different developmental stages investigated in this study. The expression level was nearly negligible in the spent stage, followed by a slight increase in the proliferative stage. However, the expression was significantly higher (p < 0.05) in the ripening stage. Afterward, there was a significant decrease in Hdh-HGAP expression in the degenerative stage (Fig. 8).Fig. 8.Hdh-HGAP mRNA expression level at different gonadal development stages in Pacific abalone. SS, spent stage; PS, proliferative stage; RS, ripening stage; DS, degenerative stage; ns, non-significant; *significant; ***highly significant.In vivo Effects of GnRH Peptide on the Gonad Development and Expression of HGAPThe administration of GnRH peptide enhanced the gonad development process compared to the control treatment in Pacific abalone. Significant expression changes were observed in the CG, TES, and OVR tissues of male and female Pacific abalone in a time-dependent manner. Although the nature of expression for both GnRH and HGAP was similar, differences in expression were observed depending on time, organs, and sex. In male Pacific abalone, the expression of both GnRH and HGAP showed increasing trends until 30 days post-peptide injection, after which it decreased. Significantly higher expression was observed at 30 days post-peptide injection, followed by a subsequent decrease in both CG and TES tissue (Fig. 9a, c). However, the expression of GnRH was higher in CG compared to TES, while HGAP showed higher levels in TES compared to OVR. In contrast, a significant increase in expression was found at 15 days post-peptide administration in female Pacific abalone (Fig. 9b, d), which gradually decreased in a time-dependent manner. At the end of the experiment, the expression was similar to the control. In female Pacific abalone, the expression of GnRH was higher in CG compared to OVR, while HGAP showed comparatively higher levels in OVR than CG.Fig. 9.Relative mRNA expression levels of GnRH and HGAP in the CG, TES and OVR tissue of H. discus hannai after GnRH peptide treatment. a Male CG. b Female CG. c TES. d OVR. ns, non-significant; *significant; ***highly significant.Hdh-HGAP Expression during Muscle Remodeling of Pacific AbaloneExpression analysis of muscle-injured Pacific abalone revealed a significant (p < 0.05) upregulation of Hdh-HGAP mRNA from the first day of injury to 6 days (Fig. 10). After the sixth day, the expression of Hdh-HGAP negligibly decreased at 12 days. Subsequently, there was a rapid reduction in its expression at 24 days. The expression levels in the control and remodeled abalone were not significantly different. However, throughout the remodeling period, the expression of Hdh-HGAP remained higher compared to the control and remodeled abalones.Fig. 10.Expression of Hdh-HGAP in muscle-injured Pacific abalone H. discus hannai. ns, non-significant; *significant; ***highly significant.Expression of Hdh-HGAP during Starvation of Pacific AbaloneSignificant differences were observed in the mRNA expression of Hdh-HGAP in Pacific abalone during nutritional stress (starvation). The expression level gradually decreased as the starvation period increased, with the lowest expression observed in the third week (Fig. 11). However, after refeeding, a significantly higher expression of Hdh-HGAP was observed compared to the starvation period and control condition.Fig. 11.Expression of Hdh-HGAP in muscle tissue of starved Pacific abalone H. discus hannai. 1 W, 1 week; 2 W, 2 weeks; 3 W, 3 weeks; RF, refeeding. ns, non-significant; *significant; ***highly significant.DiscussionThe present study successfully identified the complete sequence of the neuropeptide Hdh-HGAP in Pacific abalone. Hdh-HGAP is a cysteine-rich protein without any distinct domains or signature motifs, except for a signal peptide at its N-terminal region. The presence of a single PKC phosphorylation site in the amino acid sequence suggests its role in regulating cell proliferation and gene expression [23]. The N-myristoylation site in the Hdh-HGAP sequence may play a crucial role in signaling and response to environmental stress [24]. The predicted signal peptide and cleavage site of Hdh-HGAP align with the previously published Has-HGAP amino acid sequence, although there are some differences in the amino acid residues [18]. The phosphate (PO4) ligand within the protein structure is important for phosphorylation and dephosphorylation processes in living organisms [25]. Amino acid sequence analysis revealed that Hdh-HGAP shares more than 80% similarity with previously identified HGAP sequences from H. asinina (Has-HGAP) and H. madaka (Hma-HGAP). Due to the species-specific (abalone) nature of HGAP, comparison with other species was not possible during phylogenetic analysis. However, among the available amino acid sequences in Haliotis species, Hdh-HGAP clustered with the uncharacterized HGAP sequence of H. rufescens and two sequences of H. rubra. The other cluster consisted of two characterized sequences (H. asinina and H. madaka) and two uncharacterized sequences (H. rufescens and H. rubra). The difference between these clusters is based on the percentage of protein identity, whereas the number of amino acid residues (96) remains constant for this neuropeptide.Despite being a neuropeptide, Hdh-HGAP exhibited significant expression in muscle tissue during tissue-specific expression analysis. It is important to note that the activity of a gene is not limited to the tissue in which it is secreted, and the expression of a gene in the endocrine system can have phenotypic consequences in other tissues due to its secretory function [26]. A previous study on the growth regulatory neuropeptide of H. asinina Has-HGAP also reported similar expression patterns, with higher expression in muscle and mantle compared to secretory organs such as cerebral and pleuropedal ganglia and no expression in gills [18]. Consistent with these findings, the present study found the lowest expression of Hdh-HGAP in gill tissue. Although Hdh-HGAP is known to be involved in growth and reproductive functions, its expression in the gonad (TES and OVR) was comparatively low. It is worth mentioning that the tissue samples for tissue-specific expression analysis were collected during the spent/resting stage of gonadal development, which could explain the lower expression in the gonad. A gene is expressed when it is needed in the cells to take part in a certain function or mechanism [27].During early embryonic and larval development, Hdh-HGAP exhibited minimal expression during cell division stages (2-cell, 4-cell, morula), while higher expression was observed in trochophore, veliger, and juvenile stages compared to the control (unfertilized egg). As this is the first report of Hdh-HGAP expression during the metamorphosis of Pacific abalone, the exact reasons for these expression patterns are not fully understood. However, it is known that significant morphological and behavioral changes occur during the trochophore and veliger stages, including the formation of cilia, free swimming, retractor muscle development, mantle and foot mass formation, and shell development [28]. Therefore, it is plausible that Hdh-HGAP may have direct or indirect involvement in these developmental events. In fact, previous studies have reported the involvement of secreted growth-related genes in the metamorphosis of Pacific abalone [22].In the present study, rapidly growing juvenile Pacific abalone exhibited higher Hdh-HGAP expression compared to their stunted growth, minimum growth, and normal growth counterparts. Similar observations were made in H. asinina, where Has-HGAP was extensively expressed in fast-growing abalone compared to slow-growing abalone [18]. By comparing these findings with the known role of ganglionic neuropeptides in growth regulation in other gastropods [29‒31], we concluded that the expression of Hdh-HGAP reflects the physiological state of Pacific abalone. However, the underlying mechanism is currently unknown.In general, reproductive neuropeptides are remarkably expressed during reproductive season. The expression can vary based on sex during cell functioning [32]. The higher expression in the TES compared to the OVR suggests that Hdh-HGAP may play a more prominent role in regulating male reproductive activity than in females in Pacific abalone. Similar study reported that Has-HGAP controls the reproductive activity of male and female H. asinina, where male expression was comparatively higher [11]. The expression level of Hdh-HGAP was relatively lower compared to other reproductive regulatory neuropeptides such as Has-APGWamide, Has-Myomodulin, and Has-Whitnin. Nonetheless, a recent transcriptome and peptidome analysis in neural ganglia related to sexual maturation did not detect HGAP in Pacific abalone [10].Peptide administration influences gonad development and enhances the expression of both GnRH and HGAP in male and female Pacific abalones. In a previous study, Rajib et al. [32] reported that GnRH is responsible for the gonadal cell proliferation of both male and female Pacific abalone. Typically, hormones responsible for regulating a particular function exhibit analogous expression patterns, influenced by developmental stages, timing, and dosages (when external substances are administered) [32]. The current study reports that HGAP expression exhibits a pattern similar to GnRH in a time-dependent manner when synthetic GnRH peptide is injected. This phenomenon indicates that HGAP possibly participates in the gonad development of Pacific abalone as like other reproductive neuropeptide. While in H. asinina, there was a negligible difference in HGAP expression between the male and female anterior ganglion (CG) [11], in the case of Pacific abalone, alterations in the expression of Hdh-HGAP are noticeable depending on the sex and organs.During muscle tissue remodeling, qRT-PCR expression analysis showed that Hdh-HGAP was highly expressed throughout the repair process, with reduced expression observed at the end of remodeling. These findings suggest that the Hdh-HGAP neuropeptide may be involved in tissue remodeling following damage. Although the involvement of neuropeptides in tissue remodeling is not well studied, previous research has shown that certain neuropeptides directly or indirectly regulate tissue repair [33]. For example, Substance-P has been found to directly induce tissue repair by activating the PI3K/Akt/mTOR pathway even in the presence of IFNγ [34]. However, the receptor for HGAP and its regulatory pathway have not yet been identified, and therefore the specific mechanisms through which Hdh-HGAP participates in tissue remodeling in Pacific abalone remain unknown.Starvation was found to affect the activity of the Hdh-HGAP neuropeptide gene. During the early stages of starvation, the expression of Hdh-HGAP gradually decreased with increasing starvation duration, and a significant increase in expression was observed after refeeding. These observations suggest that starvation may negatively regulate the expression of Hdh-HGAP in Pacific abalone. During starvation, abalone are deprived of proper food and nutrients, which can potentially impact the expression of Hdh-HGAP. Although the effects of starvation on neuropeptide activity have been extensively studied [35‒37], our study is the first to report on the effects of starvation on Hdh-HGAP neuropeptide expression. Previous research found upregulated expression of HGAP in the neural ganglia of well-nourished H. asinina individuals compared to unfed individuals [18].ConclusionThe present study provides important insights into the identification and characterization of the HGAP neuropeptide in Pacific abalone, revealing its significant expression differences in various physiological processes. The inheritance of growth rate and the known role of neurosecretory cells in controlling growth in other mollusks suggest a potential involvement of Hdh-HGAP in promoting growth. The differential expression of Hdh-HGAP in different reproductive stages and peptide administration implies its potential role in regulating reproduction. The observed differential expression during metamorphosis highlights its regulatory role in this critical developmental process. Moreover, the upregulation of mRNA expression during muscle tissue remodeling and its downregulation during starvation suggest that Hdh-HGAP may play a role in tissue repair and that its expression is negatively regulated by starvation. Collectively, these findings suggest that Hdh-HGAP plays a multifaceted role in regulating growth, reproduction, metamorphosis, tissue damage repair, and the response to starvation in Pacific abalone.Statement of EthicsThe experimental protocols used in this study were approved by the Animal Care and Use Committee of Chonnam National University (approval number: CNU IACUC-YS-2020-5). The present study was conducted following the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health.Conflict of Interest StatementThe authors have no conflicts of interest to disclose.Funding SourcesThis research was supported by a grant (20180375) funded by the Ministry of Oceans and Fisheries, South Korea.Author ContributionsConceptualization, investigation, and writing-original draft preparation: K.H.K. and M.A.H.; methodology: M.A.H., S.H., D.H.C., and K.H.K.; formal analysis: M.A.H.; visualization and data curation: M.A.H. and S.H.; and validation, resources, writing-review and editing, project administration, funding acquisition, and supervision: K.H.K. All authors read and agreed to the published version of the manuscript.

Journal

Neuroendocrinology – Karger

Published: Jan 1, 2023

Keywords: Neuropeptides; Ganglia; Growth; Reproduction; Pacific abalone; Stress response

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