Pacific Abalone Research Articles

Hemolymph and transcriptome analysis to understand innate immune responses to hypoxia in Pacific abalone

Hypoxia was thought to inhibit immune responses, causing severe mortality to marine organisms. The Pacific abalone, Haliotis discus hannai, is the most widely cultured abalone species in China, but suffering from “summer mortality” in which hypoxia has been one of the main reasons. The effect of hypoxia exposure on immune responses in H. discus hannai was investigated, including cellular immune response using flow cytometry and transcriptome profiles. The influence of hypoxia treatment on the total hemocyte count (THC) of H. discus hannai was rather limited but the hemocyte survival rate of abalone increased during 48 h exposure. There was an initial rise in the production of phagocytes and reactive oxygen species (ROS) shortly (3 h) after hypoxic stimulation, but finally decreased after 48 h. This indicates that hypoxia inhibited redox activity of abalone. RNA-seq studies of gill tissues also revealed immune response mechanism in abalone after 24 h of hypoxia. Totally 954 differentially expressed genes (DEGs) were detected under different degrees of deoxygenation. Though oxidation-reduction turbulence was a result of both up- and down-regulated DEGs, cell death/apoptosis induction resulted from up-regulated DEGs, whilst DNA metabolic and immunity suppression resulted from down-regulated DEGs. In summary, our data provides evidence that deoxygenation greatly affects abalone immunity, probably making it more vulnerable. The present study also lays the foundation for further research in hypoxia-associated conditions in abalone aquaculture.

Reproductive success of released hatchery stocks in the Pacific abalone Haliotis discus hannai

There has been grave concern in stock enhancement programs regarding the effects of stocking practices on resource augmentation of ensuing generations through reproduction of released hatchery stocks. Evaluation of whether released seedlings successfully produce offspring in the natural environment is a first step toward a better understanding of such inter-generation stocking effects. Against this background, we examined the reproductive success of released hatchery stocks of a Pacific abalone species, Haliotis discus hannai, on the basis of microsatellite-based parentage analysis. Adults of hatchery-raised abalone were released from 2014 to 2016 into the Toyoura area of Funka Bay (Hokkaido Island, Japan), where natural gene flow from other habitats was expected to rarely occur. After the reproductive season of H. discus hannai in Funka Bay in 2016, we collected 2016-year-class wild-born juveniles from Toyoura and determined parentages between the juveniles and released individuals. The results of this analysis revealed that approximately 10% of the juveniles were descended from the released hatchery stocks.

An Annotated Genome for Haliotis rufescens (Red Abalone) and Resequenced Green, Pink, Pinto, Black, and White Abalone Species.

Abalone are one of the few marine taxa where aquaculture production dominates the global market as a result of increasing demand and declining natural stocks from overexploitation and disease. To better understand abalone biology, aid in conservation efforts for endangered abalone species, and gain insight into sustainable aquaculture, we created a draft genome of the red abalone (Haliotis rufescens). The approach to this genome draft included initial assembly using raw Illumina and PacBio sequencing data with MaSuRCA, before scaffolding using sequencing data generated from Chicago library preparations with HiRise2. This assembly approach resulted in 8,371 scaffolds and total length of 1.498 Gb; the N50 was 1.895 Mb, and the longest scaffold was 13.2 Mb. Gene models were predicted, using MAKER2, from RNA-Seq data and all related expressed sequence tags and proteins from NCBI; this resulted in 57,785 genes with an average length of 8,255 bp. In addition, single nucleotide polymorphisms were called on Illumina short-sequencing reads from five other eastern Pacific abalone species: the green (H. fulgens), pink (H. corrugata), pinto (H. kamtschatkana), black (H. cracherodii), and white (H. sorenseni) abalone. Phylogenetic relationships largely follow patterns detected by previous studies based on 1,784,991 high-quality single nucleotide polymorphisms. Among the six abalone species examined, the endangered white abalone appears to harbor the lowest levels of heterozygosity. This draft genome assembly and the sequencing data provide a foundation for genome-enabled aquaculture improvement for red abalone, and for genome-guided conservation efforts for the other five species and, in particular, for the endangered white and black abalone.

Different Transcriptomic Responses to Thermal Stress in Heat-Tolerant and Heat-Sensitive Pacific Abalones Indicated by Cardiac Performance.

The Pacific abalone Haliotis discus hannai is one of the most economically important mollusks in China. Even though it has been farmed in southern China for almost 20 years, summer mortality remains the most challengeable problem for Pacific abalone aquaculture recently. Here, we determined the different heat tolerance ability for five selective lines of H. discus hannai by measuring the cardiac performance and Arrhenius breakpoint temperature (ABT). The Red line (RL) and Yangxia line (YL) were determined as the most heat-sensitive and most heat-tolerant line, respectively. Heart rates for RL were significantly lower than those of the YL at the same temperature (p < 0.05). The differentially expressed genes (DEGs), which were enriched in several pathways including cardiac muscle contraction, glutathione metabolism and oxidative phosphorylation, were identified between RL and YL at control temperature (20°C) and heat stress temperature (28.5°C, the ABT of the RL) by RNA-seq method. In the RL, 3370 DEGs were identified between the control and the heat-stress temperature, while only 1351 DEGs were identified in YL between these two temperature tests. Most of these DEGs were enriched in the pathways such as protein processing in endoplasmic reticulum, nucleotide binding and oligomerization domain (NOD) like receptor signaling, and ubiquitin mediated proteolysis. Notably, the most heat-tolerant line YL used an effective heat-protection strategy based on moderate transcriptional changes and regulation on the expression of key genes.

Characterization of the complete mitochondrial genome of Pacific abalone Haliotis discus hannai

The complete mitochondrial genome of Pacific abalone Haliotis discus hannai, an economically valuable shellfish in China, was sequenced and analyzed. The mitogenome was 16,716 bp in length and contained 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes, and a putative control region. The gene arrangement and orientation were identical with other Haliotis species. Phylogenetic tree was constructed based on 13 PCGs of 7 Haliotidae species to assess their phylogenetic relationship, the tree revealed that H. discus hannai was more closely to H. rufescens and H. iris.

Identification and characteristics of muscle growth-related microRNA in the Pacific abalone, Haliotis discus hannai

BackgroundThe Pacific abalone, Haliotis discus hannai, is the most important cultivated abalone in China. Improving abalone muscle growth and increasing the rate of growth are important genetic improvement programs in this industry. MicroRNAs are important small noncoding RNA molecules that regulate post-transcription gene expression. However, no miRNAs have been reported to regulate muscle growth in H. discus hannai.Resultswe profiled six small RNA libraries for three large abalone individuals (L_HD group) and three small individuals (S_HD group) using RNA sequencing technology. A total of 205 miRNAs, including 200 novel and 5 known miRNAs, were identified. In the L_HD group, 3 miRNAs were up-regulated and 7 were down-regulated compared to the S_HD specimens. Bioinformatics analysis of miRNA target genes revealed that miRNAs participated in the regulation of cellular metabolic processes, the regulation of biological processes, the Wnt signaling pathway, ECM-receptor interaction, and the MAPK signaling pathway, which are associated with regulating growth. Bone morphogenetic protein 7 (BMP7) was verified as a target gene of hdh-miR-1984 by a luciferase reporter assay and we examined the expression pattern in different developmental stages.ConclusionThis is the first study to demonstrate that miRNAs are related to the muscle growth of H. discus hannai. This information could be used to study the mechanisms of abalone muscle growth. These DE-miRNAs may be useful as molecular markers for functional genomics and breeding research in abalone and closely related species.

A biologging technique for monitoring the egg-releasing behavior of Pacific abalone Haliotis discus hannai in the wild

A biologging technique for monitoring the egg-releasing behavior of Pacific abalone Haliotis discus hannai in the wild

Expression profiling of lncRNAs and mRNAs reveals regulation of muscle growth in the Pacific abalone, Haliotis discus hannai

Long non-coding RNAs (lncRNAs) are known to play a major role in the epigenetic regulation of muscle development. Unfortunately there is little understanding of the mechanisms with which they regulate muscle growth in abalone. Therefore, we used RNA-seq to study the muscle transcriptomes of six Haliotis discus hannai specimens: three large (L_HD group) and three small (S_HD group). We identified 2463 lncRNAs in abalone muscle belonging to two subtypes: 160 anti-sense lncRNAs and 2303 intergenic lncRNAs (lincRNAs). In the L_HD group, we identified 204 significantly differentially expressed lncRNAs (55 upregulated and 149 downregulated), and 2268 significantly differentially expressed mRNAs (994 upregulated and 1274 downregulated), as compared to the S_HD group. The bioinformatics analysis indicated that lncRNAs were relate to cell growth, regulation of growth, MAPK signaling pathway, TGF-β signaling pathway, PI3K-Akt and insulin signaling pathway, which involved in regulating muscle growth. These findings contribute to understanding the possible regulatory mechanisms of muscle growth in Pacific abalone.

Sub-optimal or reduction in temperature and salinity decrease antioxidant activity and cellularity in the hemolymph of the Pacific abalone (Haliotis discus hannai)

This study investigated the oxidative stress and hemocyte responses of Pacific abalone exposed to various water temperatures (4, 6, 8, and 10 °C) and salinities (26, 30, and 34 psu) for 7 days, to identify their tolerance ranges of temperature and salinity. The survival rate of Pacific abalone ranged from 98.7 to 100% at 8 °C and 10 °C, but dropped to 25–55% at 4 °C at all levels of salinity. The levels of superoxide dismutase and glutathione in the hemolymph were significantly higher at 4 °C and 6 °C than in the controls in all salinity groups, indicating that these temperatures induced greater stress in the Pacific abalone. Total hemocyte count was lowest at 6 °C in the 26 psu group. The percentages of apoptotic and necrotic cells were higher in the 26 psu group than in the other salinity groups, and higher in the 4 °C and 6 °C groups than in the other temperature groups. These results indicate that the lowest tolerance to water temperature and salinity in the Pacific abalone was 8 °C and 30 psu, respectively.

Microbial community and functions associated with digestion of algal polysaccharides in the visceral tract of Haliotis discus hannai: Insights from metagenome and metatranscriptome analysis.

Haliotis discus hannai, a species of Pacific abalone, is a highly valuable food source throughout Northeast Asia. As H. discus hannai primarily feed on brown algae and largely extract their energy from algal polysaccharides, understanding the mechanisms by which they digest algal polysaccharides is essential for elucidating their energy metabolism. Gut microbes, as well as the host animal, are involved in the process of polysaccharide degradation. To identify algal polysaccharide-digestion mechanisms and their origin, we analyzed the metagenome and metatranscriptome of abalone visceral extracts. Microbial communities were characterized using the 16S rRNA gene sequences in the metagenome and our results differed significantly from those of previous studies using traditional microbiological methods such as bacterial cultivation and cloning. A greater diversity of bacterial taxa was identified here than was previously identified using cultivation methods. Furthermore, the most abundant bacterial taxa also differed from previous studies, which is not common when comparing the results of bacterial culturing with those of molecular methodologies. Based on the metatranscriptome, overall functions were identified and additional analyses were performed on the coding sequences of algal polysaccharide-digestive enzymes, including alginate lyase. Results of the transcriptomic analyses suggest that alginate lyase in the visceral extracts of H. discus hannai was produced by the host itself, not by visceral bacteria. This is the first next-generation sequencing study performed on abalone to characterize the visceral microbiota and the source of the ability to digest algal polysaccharides by analyzing the metagenome and metatranscriptome together.

Nucleus-translocated matrix metalloprotease 1 regulates innate immune response in Pacific abalone (Haliotis discus hannai)

As an important economical shellfish in coastal area of China, abalone is susceptible to bacterial infection, especially Vibiro parahemolyticus (V. parahemolyticus). Matrix metalloproteinases (MMPs) have been extensively investigated in the immune response of mammals. However, little is known about the involvement of MMP in abalone innate immune system against pathogen infection. In this study, the role of MMP-1 in the immune response of Pacific abalone (Haliotis discus hannai) was explored. The results showed that V. parahemolyticus infection induced significantly elevated expression of MMP-1 as well as immune related genes including allograft inflammatory factor 1 (AIF-1), macrophage expressed gene 1 (MPEG-1) and TPA-inducible sequence 11 family protein (Tis11FP). Notably, silencing of MMP-1 reduced the expression of these genes, suggesting that MMP-1 was an upstream regulatory factor in V. parahemolyticus infection. Further analysis showed that MMP-1 was engaged in the regulation of cellular (phagocytosis, apoptosis) and humoral [superoxide dismutase (SOD), alkaline phosphatase (ALP), acid phosphatase (ACP)] immunity. Interestingly, the extracellularly distributed MMP-1 could be translocated to the nuclei of hemocytes, thereby functioning as a transcriptional regulator or by selectively activating or inactivating other components through proteolysis. Hence, our study established an important role of MMP-1 in abalone innate immunity against V. parahemolyticus infection and it represented the first report on the investigation of MMP in abalone.

Expression of Na+/K+-ATPase Was Affected by Salinity Change in Pacific abalone Haliotis discus hannai.

Na+/K+-ATPase (NKA) belongs to the P-type ATPase family, whose members are located in the cell membrane and are distributed in diverse tissues and cells. The main function of the NKA is to regulate osmotic pressure. To better understand the role of NKA in osmoregulation, we first cloned and characterized the full-length cDNAs of NKA α subunit and β subunit from Pacific abalone Haliotis discus hannai in the current study. The predicted protein sequence of the NKA α subunit, as the catalytic subunit, was well conserved. In contrast, the protein sequence of the β subunit had low similarity with those of other species. Phylogenetic analysis revealed that both the α and β subunits of the NKA protein of Pacific abalone were clustered with those of the Gastropoda. Then, the relationship between salinity changes and the NKA was investigated. Sudden salinity changes (with low-salinity seawater (LSW) or high-salinity seawater (HSW)) led to clear changes in ion concentration (Na+ and K+) in hemolymph; however, the relative stability of ion concentrations in tissue revealed that Pacific abalone has a strong osmotic pressure regulation ability when faced with these salinity changes. Meanwhile, the expression and activity of the NKA was significantly decreased (in LSW group) or increased (in HSW group) during the ion concentration re-establishing stages, which was consistent with the coordinated regulation of ion concentration in hemolymph. Moreover, a positive correlation between cyclic adenosine monophosphate (cAMP) concentrations and NKA mRNA expression (NKA activity) was observed in mantle and gill. Therefore, the sudden salinity changes may affect NKA transcription activation, translation and enzyme activity via a cAMP-mediated pathway.

Multiple calmodulin genes of the Pacific abalone, Haliotis discus hannai (Mollusca: Vetigastropoda: Haliotidae)

ABSTRACTIn this study, we identified four canonical calmodulin genes in the Pacific abalone, Haliotis discus hannai. Their full-length cDNAs were variable in the 5′ and 3′ untranslated regions, but highly similar (91–97%) in the coding region. Each of the genes encoded 149 amino acids, with 93–97% similarity among themselves and 94–98% similarity with human CAM I. There were 54 substitutions distributed unevenly throughout the coding regions, found mostly in the third codon position. Gene structure analysis revealed that each of the calmodulin genes comprised five exons and four introns. The intron positions and phases were identical and there were no introns in the fourth exon. The corresponding introns differed in their sequences and sizes. Expression profiles of nine tissues from abalone revealed that the calmodulin genes were transcribed in common in gill and mantle tissue, but differentially in the other tissues. A phylogenetic analysis based on the amino acid sequences revealed that calmodulin C was the most common isoform in Gastropoda and calmodulin was the most diverged isoform. An in silico analysis of the calmodulin genes identified paralogous genes in other Haliotis species, indicating that gene duplication might have occurred in the last common ancestor of Haliotis.Abbreviations: ORF: open reading frame; RACE: random amplification of cDNA end; TSA: transcriptome shotgun assembly; UTR: untranslated region

A comparison of offspring growth and survival among a wild and a selected strain of the Pacific abalone (Haliotis discus hannai) and their hybrids

The commercial breeding and cultivation of the Pacific abalone (Haliotis discus hannai) in China have experienced remarkable progress over the last three decades. Both genetic breeding programs and improvements in cultivation techniques have contributed to this advancement. However, no published data have ever distinguished the progress contributed by genetic breeding from the gross advancement. We compared the offspring growth and survival among a wild and a selected strain (AO) of the Pacific abalone (Haliotis discus hannai) and their hybrids (wild♀ × AO♂) and tried to distinguish the advancement provided by genetic breeding. The offspring metamorphosis rate, growth and survival of the selected strain of the Pacific abalone were superior to those of the wild strain. In the artificial diet feeding phase, the offspring shell growth and survival rates of the selected strain were >17% and 30% higher than those of the wild stain, respectively. In the overwintering phase, the offspring shell growth, wet weight growth and survival rates were >28%, 50% and 35% higher than those of the wild stain, respectively. Although the oocytes were from the same source, the hybrids showed a superior performance compared to the purebred wild strain, especially in the metamorphosis phase, which also indicated the progress made by the selected strain in adapting to the culturing conditions. Our results demonstrated that remarkable genetic improvement has been made and that the selected strain is much more suitable for commercial abalone breeding and cultivation in China.

Sulfated polysaccharides from pacific abalone reduce diet-induced obesity by modulating the gut microbiota

Sulfated polysaccharides from abalone (Haliotis discus hannai Ino) gonad (AGSP) is shown to have various bioactivities, but it remains unknown whether AGSP produces any effect on high fat diet (HFD)-induced obesity. This study indicated that AGSP inhibited weight gain and improved body composition in HFD-fed mice without reducing food consumption. Moreover, AGSP reduced fat accumulation in the liver and epididymal fat tissues of HFD-fed mice via the modulation of lipid metabolism. Such effects could be associated with the improvement of the gut microbiota as indicated by a decreased ratio of Firmicutes/Bacteroidetes and overall structure changes of the gut microbiota. Butyrate as the end product of bacterial fermentation was significantly increased in AGSP-fed mice, and thus may inhibit lipid-related gene expressions via G protein-coupled receptors-dependent pathways. In short, it suggested that AGSP can be used as beneficial polysaccharides to improve obesity and its associated disorders.

Expression profile and reproductive regulation of APGWamide in Pacific abalone (Haliotis discus hannai)

Neuropeptides in the central nervous system regulate reproductive activities in vertebrates. Ala-Pro-Gly-Trp-NH2 (APGWamide), a neuromediator expressed in the neural ganglia of mollusks, controls sexual maturation and reproduction. To clarify the role of APGWamide in sexual behavior regulation and gamete cell maturation in mollusks, we cloned the cDNA of APGWamide precursor (Hdh-APGWamide) and examined the spatiotemporal expression of the transcript in the Pacific abalone Haliotis discus hannai. The 222-amino acid sequence of the precursor deduced from the cDNA sequence showed typical features of gastropod APGWamide precursors. Phylogenetic analysis revealed that Hdh-APGWamide is classified with other gastropod APGWamide precursors, which form a separate branch from those of the bivalves. Hdh-APGWamide mRNA was highly expressed in the neural ganglia in both sexes. In females, the three ganglia (pleuro-pedal ganglion, PPG; branchial ganglion, and cerebral ganglion) showed similar expression in immature and mature animals, whereas in males, the level in the PPG only was higher at maturity (P < 0.05). In vivo injection of APGWamide or 5-hydroxytryptamine (10−3 M) increased the frequency of spawning and the number of released sperm cells by mature males (P < 0.05), while concentrations above 10−7 M enhanced germinal vesicle breakdown in fully developed cultured oocytes (P < 0.05). Thus, the phylogenetic branch of the APGWamide precursor gene in Haliotidae was separate from the other branches under the phylum Mollusca, and this gene exhibited ganglion-specific expression, indicating that it may induce final maturation and spawning in both sexes of Haliotis spp.

Gonadal Transcriptome Analysis of Pacific Abalone Haliotis discus discus: Identification of Genes Involved in Germ Cell Development.

Little is known about the molecular mechanisms governing gonadal developmental processes in abalones. Here, we conducted transcriptome analysis of Pacific abalone Haliotis discus discus for gene discovery in the brain, ovary, testis, and unfertilized eggs. Among the annotated unigenes, 48.6% of unigenes were identified by Venn diagram analysis as having universal or tissue-specific expression. Twenty-three genes with gonad-biased gene ontology (GO) terms were first obtained. Secondly, 36 genes were found by screening known gene names related to germ cell development. Finally, 17 genes were obtained by querying the annotated unigene database for zygotically expressed gonadal genes (ovary and testis) and maternally expressed gonadal genes (ovary, testis, and unfertilized eggs) using keywords related to reproduction. To further verify tissue distribution pattern and subcellular localization of these genes, RT-PCR and in situ hybridization were performed using a unigene encoding a germ cell marker, vasa, as control. The results showed that vasa was expressed mainly in the early developmental stages of germ cells in both sexes. One of the candidate genes, vitelline envelope zona pellucida domain protein 12 (ZP12), was expressed in the primordial germ cells of immature gonad and early developmental stages of germ cells of the adult female. The results obtained from the present study suggest that vasa and ZP12 are involved in germ cell development of Pacific abalone and that ZP12 is an especially useful germ cell-specific marker in immature adults. The current gonadal transcriptome profile is an extensive resource for future reproductive molecular biology studies of this species.

북방전복 (Haliotis discus hannai) 의 열 충격 결합 단백질1 (HspBP1, Heat shock binding protein1)유전자 동정 및 발현분석

북방전복 (Haliotis discus hannai) 의 열 충격 결합 단백질1 (HspBP1, Heat shock binding protein1)유전자 동정 및 발현분석

The detrimental effects of CO2-driven chronic acidification on juvenile Pacific abalone (Haliotis discus hannai)

To test the effects of chronic stress caused by CO2-driven decreases in pH (NBS scale) on molluscs, juvenile Pacific abalone (Haliotis discus hannai), an economically important gastropod, were cultured at pH 8.1 (control), pH 7.9 or 7.7 for 3 months. Eroded shell surfaces, reduced growth rates, and altered biochemical composition and energy metabolism were found in abalone cultured in acidified conditions. Glycogen contents and crude lipid contents were significantly higher in the muscle of abalone from the control treatments than in those from the pH 7.7 treatment, indicating that allocation of tissue energy storage was altered by chronic acidification. The ratio of the oxygen consumption rate to the ammonia excretion rate (O:N ratio) of individuals cultured at two acidified treatments decreased significantly compared with the controls, suggesting a decreased use of body lipid and carbohydrates as a metabolic substrate under chronic acidified conditions. Chronic acidification had detrimental effects on the physiological activity of the Pacific abalone; however, the ability to acclimate varied among individuals. A minority of tested animals incubated at acidified conditions exhibited undiminished performance. The Pacific abalone may face severe challenges in the near future, but they may also hold considerable ability to acclimate to ocean acidification.

니켈 (NiCl₂) 및 수온 스트레스에 의한 북방전복, Haliotis discus hannai (Ino, 1953) 의 생리학적 변화

니켈 (NiCl₂) 및 수온 스트레스에 의한 북방전복, Haliotis discus hannai (Ino, 1953) 의 생리학적 변화