Pacific Abalone Research Articles

Gluconeogenesis and glycogen metabolism during development of Pacific abalone, Haliotis discus hannai.

In lecithotrophic larvae, egg yolk nutrients are essential for development. Although yolk proteins and lipids are the major nutrient sources for most animal embryos and larvae, the contribution of carbohydrates to development has been less understood. In this study, we assessed glucose and glycogen metabolism in developing Pacific abalone, a marine gastropod mollusc caught and cultured in east Asia. We found that glucose and glycogen content gradually elevated in developing abalone larvae, and coincident expression increases of gluconeogenic genes and glycogen synthase suggested abalone larvae had activated gluconeogenesis and glycogenesis during this stage. At settling, however, glycogen sharply decreased, with concomitant increases in glucose content and expression of Pyg and G6pc, suggesting the settling larvae had enhanced glycogen conversion to glucose. A liquid chromatography-mass spectrometry (LC/MS)-based metabolomic approach that detected intermediates of these pathways further supported active metabolism of glycogen. Immunofluorescence staining and in situ hybridization suggested the digestive gland has an important role as glycogen storage tissue during settlement, while many other tissues also showed a capacity to metabolize glycogen. Finally, inhibition of glycolysis affected survival of the settling veliger larvae, revealing that glucose is, indeed, an important nutrient source in settling larvae. Our results suggest glucose and glycogen are required for proper energy balance in developing abalone and especially impact survival during settling.

Molecular cloning, in silico characterization and expression analysis of axonemal protein 66.0 in Pacific abalone, Haliotis discus hannai

Axonemal protein 66.0 is one of the major axonemal dynein protein involved in activation of sperm motility. The axonemal protein 66.0 gene was successfully cloned and characterized from testis tissue in the Pacific abalone, Haliotis discus hannai. The cloned gene was named Hdh-Axp66.0 based on the predicted molecular mass. The full-length cDNA of Hdh-Axp66.0 was 2023 bp, with a coding region of 1694 bp; it encoded 564 deduced amino acids with a predicted molecular mass of 65.42 kDa and an isoelectric point of 8.68. In silico analysis prediction suggested this gene has two cAMP-dependent protein kinase phosphorylation sites, seven predicted protein kinase C phosphorylation sites, three N-linked glycosylation sites, a tyrosine kinase phosphorylation motif, three coiled-coil domains, and several Ca2+ binding sites, which revealed the possible involvement of Hdh-Axp66.0 in activation of sperm motility. Tissue distribution analysis indicated that Hdh-Axp66.0 was distributed in testis, heart, gill and mantle tissues. However, quantitative real-time PCR (qPCR) revealed that Hdh-Axp66.0 mRNA was highly expressed in testis tissue. Further, highly elevated relative mRNA expression was observed in testis tissue during the ripening stage compared with different testicular developmental stages. These results suggest the possible involvement of Hdh-Axp66.0 in testicular development and reproduction.

In silico prediction of neuropeptides from the neural ganglia of Pacific abalone Haliotis discus hannai (Mollusca: Gastropoda)

Neuropeptides play a central role in the regulation of reproduction, growth, development, and various other physiological processes. In our study, 26 neuropeptide precursors were identified from the pleuropedal and cerebral ganglia of Pacific abalone using transcriptome analysis. Of them, two (neuromacin and neuroglian) were potentially novel and two (myomodulin and FMRFamide) were involved in the reproductive regulation of mollusks. A BLAST search indicated that most of these neuropeptides exhibited the highest homologies with neuropeptides of other protostomian species. Myomodulin, FMRFamide, and molluscan insulin-related peptides shared 87%, 72%, and 88% sequence identities with homologous peptides of the tropical gastropod mollusk, Haliotis asinina. In silico analysis revealed that these identified neuropeptide precursors were likely to be extracellular secreted proteins. Alignment of multiple cerebrin, FFamide, and insulin-related peptide sequences illustrated that most of their biologically active residues were highly conserved with other invertebrate homologous neuropeptide residues. Three-dimensional (3D) structures of adipokinetic hormone, allatostain (A, B, and C), allatotropin, cerebrin, clionin, conopressin, elevenin, and neuromycin precursors in H. discus hannai exhibited a helix-loop-helix structure. Based on phylogenetic analysis, buccalin A1 and A2, allatostain B, and FCAP neuropeptide precursors of Pacific abalone formed a clade with other gastropod and bivalve mollusks neuropeptide precursors. This study provides a novel insight for further functional studies of abalone and other gastropod mollusks.

Assessment of Suitable Reference Genes for RT-qPCR Normalization with Developmental Samples in Pacific Abalone Haliotis discus hannai

Potential utility of 14 candidate housekeeping genes as normalization reference for RT-qPCR analysis with developmental samples (fertilized eggs to late veliger larvae) in Pacific abalone Haliotis discus hannai was evaluated using four different statistical algorithms (geNorm, NormFinder, BestKeeper and comparative ΔCT method). Different algorithms identified different genes as the best candidates, and geometric mean-based final ranking from the most to the least stable expression was as follow: RPL5, RPL4, RPS18, RPL8, RPL7, UBE2, RPL7A, GAPDH, RPL36, PPIB, EF1A, ACTB and B-TU. The findings were further validated via relative quantification of metallothionein (MT) transcripts using the stable and unstable reference genes, and expression levels of MT were greatly influenced according to the choice of reference genes. In overall, our data suggest that RPL5 and RPS18, either singly or in combination, are appropriate for normalizing gene expression in developmental samples of this abalone species, whereas ACTB, B-TU and EF1A are less stable and not recommended. In addition, our findings propose that standard deviations in geometric ranking as well as geometric mean itself should also be taken into account for the final selection of reference gene(s). This study could be a useful basis to facilitate the generation of accurate and reliable RT-qPCR data with developmental samples in this abalone species.

Expression of Heat Shock Proteins in Thermally Challenged Pacific Abalone Haliotis discus hannai.

Summer mortality, caused by thermal conditions, is the biggest threat to abalone aquaculture production industries. Various measures have been taken to mitigate this issue by adjusting the environment; however, the cellular processes of Pacific abalone (Haliotis discus hannai) have been overlooked due to the paucity of genetic information. The draft genome of H. discus hannai has recently been reported, prompting exploration of the genes responsible for thermal regulation in Pacific abalone. In this study, 413 proteins were systematically annotated as members of the heat shock protein (HSP) super families, and among them 26 HSP genes from four Pacific abalone tissues (hemocytes, gill, mantle, and muscle) were differentially expressed under cold and heat stress conditions. The co-expression network revealed that HSP expression patterns were tissue-specific and similar to those of other shellfish inhabiting intertidal zones. Finally, representative HSPs were selected at random and their expression patterns were identified by RNA sequencing and validated by qRT-PCR to assess expression significance. The HSPs expressed in hemocytes were highly similar in both analyses, suggesting that hemocytes could be more reliable samples for validating thermal condition markers compared to other tissues.

Characterization of testis-specific serine/threonine kinase 1-like (TSSK1-like) gene and expression patterns in diploid and triploid Pacific abalone (Haliotis discus hannai; Gastropoda; Mollusca) males.

Testis-specific serine/threonine kinase 1-like (TSSK1-like), which plays important roles in late-phase spermatogenesis and male fertility, was characterized in Pacific abalone Haliotis discus hannai, an important commercial marine gastropod. Further, its expression patterns were assessed in diploid and induced triploid males showing differential degrees of testis maturation. Abalone TSSK1-like shared conserved structural features with mammalian TSSK1s and other potential metazoan orthologs, especially regarding the catalytic STKc domain. Phylogenetically, abalone TSSK1-like displayed a genetic affiliation with its molluscan TSSK1-like orthologs and human TSSK1. Additionally, abalone TSSK1-like gene showed a tetrapartite exon-intron organization, unlike the intronless structure of most amniotic tetrapodian TSSK1s. Molecular phylogenetic analysis in the metazoan lineage suggested a possible revision in the origin of the earliest ancestral TSSK1. Further, abalone TSSK1-like showed testis-predominant expression, which was significantly influenced by both age and seasonal reproductive cycles. Comparative expression analyses between diploid and triploid abalone males suggested that robust TSSK1-like expression occurred primarily at the post-meiotic stage. Additionally, RT-PCR assay indicates that mature abalone sperms retain TSSK1-like transcripts after release. Taken together, this study provides useful insights for further studies to assess male reproduction and sterility and/or partial fertility of induced male triploidy in abalone species.

Genetic features of Haliotis discus hannai by infection of vibrio and virus.

Haliotis discus hannai more commonly referred to as the Pacific Abalone is of significant commercial and economical value in South Korea, with it being the second largest producer in the world. Despite this significance there is a lack of genetic studies with regards to the species. Most existing studies focused mainly on environmental factors. To provide a comprehensive review describing the genetic feature of Haliotis discus hannai by infection of vibrio and virus. This review summarized the immune response in the Haliotis spp. with regards to immunological genes such as Cathepsin B, C-type lectin and Toll-like receptors. Genetic studies with regards to transposable elements and miRNAs are few and far between. A study identified LTR retrotransposon Ty3/gypsy in the species. As to miRNA, a single study identified numerous miRNAs in the Haliotis discus hannai. This paper sought to provide an overview of genetic perspective with regards to immune response genes, transposable elements and miRNAs.

Identification, characterization, and expression analysis of a serotonin receptor involved in the reproductive process of the Pacific abalone, Haliotis discus hannai.

Serotonin receptor (5-HT) is a biogenic amine acting as a neurotransmitter and neuromodulator that mediates various aspects of reproduction and gametogenesis. The full-length nucleotide sequence of Haliotis discus hannai encodes a protein of 417 amino acids with a predicted molecular mass of 46.54kDa and isoelectric point of 8.94. The structural profile of 5-HTHdh displayed key features of G protein-coupled receptors, including seven hydrophobic transmembrane domains, putative N-linked glycosylation sites, and several phosphorylation consensus motifs. It shares the highest homology of its amino acid sequence with the 5-HT receptor from Haliotis asinina, and to lesser extent of human 5-HT receptor. The cloned sequence possesses two cysteine residues (Cys-115 and Cys-193), which are likely to form a disulfide bond. Phylogenetic comparison with other known 5-HT receptor genes revealed that the 5-HTHdh is most closely related to the 5-HTHa receptor. The three-dimensional structure of the 5-HTHdh showed multiple alpha helices which is separated by a helix-loop-helix (HLH) structure. Quantitative PCR demonstrated that the receptor mRNA was predominantly expressed in the pleuropedal ganglion. Significant differences in the transcriptional activity of the 5-HTHdh gene were observed in the ovary at the ripening stage. An exclusive expression was detected in pleuropedal ganglion, testis, and ovary at higher effective accumulative temperature (1000°C). In situ hybridization showed that the 5-HTHdh expressing neurosecretory cells were distributed in the cortex of the pleuropedal ganglion. Our results suggest that 5-HTHdh synthesized in the neural ganglia may be involved in oocyte maturation and spawning of H. discus hannai.

Characterization and expression analysis of a GnRH-like peptide in the Pacific abalone, Haliotis discus hannai

Gonadotropin-releasing hormone (GnRH) is a key neuropeptide of vertebrates, involved in gonadal maturation and primarily regulated by the hypothalamic-pituitary-gonadal axis. The full length GnRH-like cDNA isolated from the cerebral ganglion of Haliotis discus hannai, encodes a deduced protein of 216 amino acids with a theoretical molecular mass of 23.36 kDa and an isoelectric point of 7.72. The GnRH transcript comprises a putative signal peptide, a GnRH dodecapeptide, a proteolytic processing site, and a GnRH associated peptide (GAP). Comparative structural analysis revealed that the cloned sequence shares relatively high homology with other molluscan species and a lower degree of amino acid identity with GnRH of piscine vertebrates. Phylogenetic comparison with other known GnRH genes revealed that the GnRH-like mRNA of H. discus hannai is most closely related to the marine gastropod, H. laevigata. Three-dimensional homology structure of H. discus hannai GnRH and GAP exhibited a helix-loop-helix structure. Quantitative PCR assay demonstrated wide expression of GnRH in all ganglia, among them cerebral ganglion exhibited the highest expression level. Significantly higher expression was observed in cerebral ganglion and gonadal tissues at higher effective accumulative temperature (1000 °C). In situ hybridization showed that the GnRH expressing neurosecretory cells distributed throughout the cortex of the cerebral ganglion. These results suggest that GnRH synthesized from the cerebral ganglia may be involved in gonadal maturation and regulating the secretion of other reproductive hormones.

Antimicrobial resistance of pathogenic Aeromonas spp. isolated from marketed Pacific abalone (Haliotis discus hannai) in Korea.

The object of this study was to identify potential health concerns of the Aeromons spp. isolated from marketed Pacific abalone (Haliotis discus hannai) with respect to their virulence and antimicrobial resistance patterns. We identified 29 strains of aeromonads consisting of five species; Aeromonas hydrophila (n=9), Aeromonas enteropelogenes (n=14), Aeromonas veronii (n=3), Aeromonas salmonicida (n=2) and Aeromonas sobria (n=1), by employing series of biochemical tests and gene sequencing. In the phenotypic virulence assays, all isolates showed gelatinase and caseinase activities, while lipase formation (69%), phospholipase production (90%), DNase formation (82%), slime production (49%) and haemolysis activity (α=18% and β=82%) were also detected among isolates. Prevalence of virulence genes; aerA (100%), fla (66%), ahyB (73%), act (52%), alt (42%), ast (35%), ser (52%), gcat (69%), ascV (43%), hlyA (83%), lip (52%) and exu (59%) were detected by PCR assays. In disc diffusion test, 100% resistance was detected against ampicillin while cephalothin, rifampicin, oxytetracycline, colistine sulphate, nalidixic acid and piperaciliin were resisted by 86, 73, 42, 35, 28, 20 and 20% of the isolates respectively. Thirteen (45%) of the isolates showed multiple antimicrobial resistance (MAR) indices ≥0·2. Our findings suggest that the potential health risk posed by the abalone-borne Aeromonas spp. should not be underestimated. This is the first time to evaluate possible public health risks upon consumption of abalone harbored Aeromonas spp. and also to isolate potential pathogenic and multidrug-resistant Aeromonas spp. from Pacific abalone in Korea.

Genetic determination of sex and shell color in the Pacific abalone Haliotis discus hannai revealed by an integrated linkage map.

Integrated linkage maps for each sex have been constructed for the Pacific abalone Haliotis discus hannai using three F1 mapping families based on co-dominant markers. A total of 273 markers were placed on the female map, spanning 927.3cM with an average interval of 3.64cM, whereas 277 markers were mapped on the male map, covering 727.0cM with an average spacing of 2.80cM. Both female and male maps consisted of 18 linkage groups, corresponding well with the number of chromosomes. Furthermore, the sex-determining locus and the green/orange shell color controlling locus were mapped to the linkage group 3 (LG3) and LG9 respectively. A marker completely linked to phenotypic sex was identified, and the sex determination system was further concluded as paternal heterogametic (males XY and females XX). Based on the segregation ratio of the shell color in the progeny, a simple recessive model of epistasis was proposed to explain the distribution of different color morphs (green, orange and blue): the recessive allele determining orange type masks the effect of the locus controlling green and blue types, whereas the dominant allele at the green/orange locus permits the expression of green and blue types controlled by another locus. The current consensus map provides a useful framework for genetic studies in the Pacific abalone. Mapping of the sex-determining locus and the shell color-controlling locus leads to further understanding of the mechanisms underlying these important traits.

Greenlip Abalone (Haliotis laevigata) Genome and Protein Analysis Provides Insights into Maturation and Spawning.

Wild abalone (Family Haliotidae) populations have been severely affected by commercial fishing, poaching, anthropogenic pollution, environment and climate changes. These issues have stimulated an increase in aquaculture production; however production growth has been slow due to a lack of genetic knowledge and resources. We have sequenced a draft genome for the commercially important temperate Australian ‘greenlip’ abalone (Haliotis laevigata, Donovan 1808) and generated 11 tissue transcriptomes from a female adult abalone. Phylogenetic analysis of the greenlip abalone with reference to the Pacific abalone (Haliotis discus hannai) indicates that these abalone species diverged approximately 71 million years ago. This study presents an in-depth analysis into the features of reproductive dysfunction, where we provide the putative biochemical messenger components (neuropeptides) that may regulate reproduction including gonad maturation and spawning. Indeed, we isolate the egg-laying hormone neuropeptide and under trial conditions induce spawning at 80% efficiency. Altogether, we provide a solid platform for further studies aimed at stimulating advances in abalone aquaculture production. The H. laevigata genome and resources are made available to the public on the abalone ‘omics website, http://abalonedb.org.

Study on Tolerance and Immune Response of Pacific Abalone Haliotis discus hannai during Simulated Air Exposure of Live Transportation

Pacific abalone (Haliotis discus hannai) is an economically important species of sea snails found in China. Live abalones sold in domestic markets are sealed in insulated containers for transportation over extended periods of time (6–24 h), during which this species may experience stressful conditions and spoil. This study assessed the survival rate and weight loss rate (WLR) of abalone under various treatments to determine the optimal packaging transport conditions. Specifically, temperature, supplemental humidity and oxygen, and air exposure during simulated live transport were investigated. Results revealed that the survival rate increased significantly after cold-water shock and humidity control, whereas oxygen supplements did not have a significant effect. In addition, WLR significantly increased over time (P < 0.05). An additional experiment was conducted to investigate the stress response caused by air exposure. Hemocyte parameters, including total hemocyte count (THC), hemocyte mortality, and reactive oxygen species (ROS), were estimated for 2, 6, 12, 24, and 30 h after air exposure at different temperatures. Results revealed that THC significantly decreased after 30 h of air exposure at 5 and 10°C, and after 24 h of air exposure at 20°C. Hemocyte mortality and ROS significantly increased, peaking after 30 h at 5 and 10°C, followed by a gradual decrease over the subsequent 24 h recovery period. THC and ROS levels remained higher than the control levels by the end of the recovery period. The parameters investigated in this study could serve as indicators for the effects on the functional immune response and physiological stress of abalone during live transportation.

Physiological Response of Pacific Abalone (Haliotis discus hannai) Hepatopancreas to Heat Stress

Pacific abalone Haliotis discus hannai is one of the most economically important marine shellfish and is mainly distributed in the cooler seas of northern China. The molecular reactions that occur in this species under high temperature conditions have not been determined. To understand the molecular mechanisms of this species under heat stress, transcriptomic analysis of the hepatopancreas was performed at 33°C (22°C was used for the control) for 1 h on an Illumina HiSeq 2500 platform (pooling of six animals into single RNA-sequencing libraries). The results showed that 17,852 unigenes were annotated. In all, 8,532 genes were significantly differentially expressed in the test group compared with the control group, including 4,788 upregulated and 3,744 downregulated genes. Enrichment analysis identified some noteworthy GO terms (cellular process and response to stimulus) and pathways (protein processing in the endoplasmic reticulum (ER) and apoptotic signaling pathways), and these were further analyzed. Analysis showed that genes indicative of mitochondrial damage were upregulated; DNA damage formation, and fatty acid and amino acid synthesis increased; fatty acid decomposition reduced; and misfolded proteins accumulated in the ER, and the unfolded protein response and ER-associated degradation were triggered to relieve ER stress. In addition, the apoptotic pathway was activated; however, some genes associated with anti-apoptosis and molecular chaperones were upregulated to maintain homeostasis after heat stress. These data provide an increased understanding of the heat stress mechanisms in Pacific abalone.

Scanning Electron Micrographs of the Early Life Stages of the Pacific Abalone Haliotis discus hannai (INO, 1953)

The Pacific abalone Haliotis discus hannai is a valuable food resource in China. Understanding the early development of this species will allow cell fate tracking and may help to guide artificial seeding. Scanning electron microscopy (SEM) was used to examine the morphologies of the major life stages of H. discus hannai from artificial fertilization to the juvenile stage (23 days postfertilization). The results represented the first comprehensive SEM micrograph record of early development in the Haliotidae. Special attention was also paid to shell formation in H. discus hannai, as these results might help inform biomineral-related research.

Identification and Function of GnRH-like Peptide in the Pacific Abalone, Haliotis discus hannai.

Gonadotropin-releasing hormone (GnRH) is an important regulator of reproductive function in various vertebrates and invertebrates. In the present study, we have identified the GnRH-like peptide cDNA and peptide from the cerebral ganglion (CG) of the Pacific abalone, Haliotis discus hannai. Pacific abalone GnRH-like peptide (hdhGnRH-like peptide) cDNA encodes precursor, which possesses the typical organization of the known mollusk GnRH-like peptide precursors, including a hydrophobic signal peptide, GnRH-like peptide, and a cleavage site followed by a GAP-like peptide region. Three hdhGnRH-like peptides, pQNYHFSNGWHAamide (hdhGnRH-11amide), pQNYHFSNGWHA (hdhGnRH-11OH), and pQNYHFSNGWHAG (hdhGnRH-12OH), were determined from the acid/acetone extract of the CG by mass spectrometry (LC-MS/MS) analysis. The hdhGnRH-like peptide mRNA expression was detected not only in the CG but also in gonads, and hdhGnRH-11amide was also detected in the extract of gonads. The mRNA expression of hdhGnRH-like peptide in the CG was lower in spawned males than in non-spawned animals, while no change in hdhGnRH-like peptide mRNA expression was shown in both ovulated and non-ovulated abalone. The hdhGnRH-11amide induces spawning and ovulation of both mature males and females in a concentration-dependent fashion following intramuscular injection. These results indicate that three hdhGnRH-like peptides are yielded from a single hdhGnRH-like peptide precursor, and that at least hdhGnRH-11amide is involved in the control of reproduction of the Pacific abalone.

An Effective Microinjection Method and TALEN-Mediated Genome Editing in Pacific Abalone.

Pacific abalone, Haliotis discus hannai, is an economically important marine mollusk species and an important model animal for studies on ecological, fertilization and developmental biology. While embryonic injection and genome editing have been wildly used in gene function study and trait improvement in many species, they have not been developed in abalones. In this study, we reported an effective method to inject exogenous materials in H. discus hannai unfertilized eggs. The injected eggs could be fertilized at a ratio of 52.6% ± 5.9% and hatch at a ratio of 14.6% ± 1.6%. On the base of this, we further developed an efficient genome editing approach in this species with the transcription activator-like effector nuclease (TALEN) technique. Two TALEN pairs targeting the coding sequence of the abalone nodal gene were assembled and tested. While one of the TALEN pairs showed no detectable mutation efficacy, the other one generated mutations in 50% of the targeted loci. The mutation includes small insertions and deletions and base pair replacements like that reported in other species when the TALEN method was applied. Overall, this is the first study to demonstrate site-specific genome editing in abalone. This work can serve as a reference for future studies focusing on the functional genomics in mollusks.

Neural Ganglia Transcriptome and Peptidome Associated with Sexual Maturation in Female Pacific Abalone (Haliotis discus hannai).

Genetic information of reproduction and growth is essential for sustainable molluscan fisheries and aquaculture management. However, there is limited knowledge regarding the reproductive activity of the commercially important Pacific abalone Haliotis discus hannai. We performed de novo transcriptome sequencing of the ganglia in sexually immature and mature female Pacific abalone to better understand the sexual maturation process and the underlying molecular mechanisms. Of the ~305 million high-quality clean reads, 76,684 transcripts were de novo-assembled with an average length of 741 bp, 28.54% of which were annotated and classified according to Gene Ontology terms. There were 256 differentially expressed genes between the immature and mature abalone. Tandem mass spectrometry analysis, as compared to the predicted-peptide database of abalone ganglia transcriptome unigenes, identified 42 neuropeptide precursors, including 29 validated by peptidomic analyses. Label-free quantification revealed differential occurrences of 18 neuropeptide families between immature and mature abalone, including achatin, FMRFamide, crustacean cardioactive peptide, and pedal peptide A and B that were significantly more frequent at the mature stage. These results represent the first significant contribution to both maturation-related transcriptomic and peptidomic resources of the Pacific abalone ganglia and provide insight into the roles of various neuropeptides in reproductive regulation in marine gastropods.

Haliotis papulata Reeve 1846 (Vetigastropoda: Haliotidae), the pimpled abalone, is the correct name for the small Indo-West Pacific abalone referred to as Haliotis thailandis Dekker &amp; Patamakanthin 2001 and not as a synonym of Haliotis varia Linnaeus 1758

The syntypes of Haliotis papulata Reeve 1846 have been re-examined and are found to not represent specimens of H. varia Linnaeus 1758. Haliotis papulata appears to be the correct name for specimens that are currently considered H. thailandis Dekker &amp; Patamakanthin 2001. The synonymy of Haliotis thailandis within H. papulata provides a more detailed understanding of the distribution of the species and its probable close phylogenetic relationships with the Indo-Pacific abalone species, Haliotis unilateralis Lamarck 1822 and H. clathrata Reeve 1846

Molecular characterization, expression analysis, and functional properties of multiple 5-hydroxytryptamine receptors in Pacific abalone (Haliotis discus hannai)

Neurotransmitters such as serotonin (5-hydroxytryptamine; 5-HT) in the central nervous system regulate diverse physiological functions, including reproduction, feeding, learning, and memory, in diverse animal phyla. 5-HT and the 5-HT1 subtype receptor play important roles in sexual maturation and in the initiation of gamete release in mollusks. However, little is known about the involvement of other 5-HT receptor subfamilies in the reproduction process. In the present study, we identified the cDNAs encoding eight subtypes of 5-HT receptors from the ganglia tissues of the Pacific abalone Haliotis discus hannai (Mollusca; Gastropoda; Haliotidae), and examined the gonadal expression of the transcripts of 5-HT receptors. A phylogenetic analysis indicated that the molluskan 5-HT receptors are largely classified into four major clades: 5-HT1/5/7, 5-HT2, 5-HT4, and 5-HT6. Among the H. discus hannai (Hdh) 5-HT1-7 transcripts, Hdh5-HT1B, 4A, 4B, and 6 were the major subtypes detected in the mature ovary. Estradiol-17β injection into the pedal sinus induced the downregulation of 5-HT4B and upregulation of 5-HT6 transcripts in the ovary of mature abalone within 72 h. In HEK293 cells overexpressing Hdh5-HT1B, forskolin-stimulated cAMP response element luciferase (CRE-Luc) reporter activity was inhibited by 5-HT in a dose-dependent manner, whereas serum response element luciferase (SRE-Luc) activity was not affected. In Hdh5-HT4A-expressing HEK293 cells, forskolin-stimulated CRE-Luc and SRE-Luc reporter activities were both marginally increased by treatment with a high dose of 5-HT. Our results provide new insights into the roles of 5-HT through diverse G protein-coupled 5-HT receptors in the reproductive process of mollusks.