Small Abalone Haliotis Diversicolor Research Articles

Abiotic Stress Tolerance of Juvenile Small Abalone Haliotis diversicolor aquatilis (Obunjagi) to Gamma Irradiation

Jwa, M.S. and Hong, C.-Y., 2023. Abiotic stress tolerance of juvenile small abalone Haliotis diversicolor aquatilis (obunjagi) to gamma irradiation. Journal of Coastal Research, 39(1), 55–62. Charlotte (North Carolina), ISSN 0749-0208. In South Korea, the small abalone (obunjagi), Haliotis diversicolor aquatilis, is a prominent commercial seafood species. However, summer circumstances frequently impede abalone productivity, emphasizing the need for suitable approaches to promote abalone viability. The purpose of this study was to see if the survival rate and physiological responses of juvenile H. diversicolor aquatilis changed after exposure to low-level 60Co-gamma irradiation under high temperature (HT) and low salinity (LS) stress conditions (20 Gy). For this research, juvenile small obunjagi, H. diversicolor aquatilis, were obtained from Jeju City fishing vendors, Korea. Before the experiment, all abalone samples were acclimated for 7 days in a seawater-filled tank at 23°C ± 0.3°C and 32 ± 0.2 psu under starvation conditions. Seventy abalones (20 male and 50 female) were gamma irradiated and 30 abalones (10 male and 20 female) were used as controls. After 6 weeks of malnutrition, abalone survivability and the quantity of cardiovascular hemocytes rose dramatically after 20-Gy exposure compared with the control (0 Gy). The viability of abalone was dramatically reduced by HT stress but not by LS stress. The initially improved immune response-mediated hormetic impact might be connected to abalone survival rate under famine and HT stress, and the hormesis-induced gamma radiation exposure strategy will successfully raise abalone productivity throughout summer.

Sex-specific markers developed by next-generation sequencing confirmed a male heterogametic sex determination in small abalone, Haliotis diversicolor

Small abalone (Haliotis diversicolor) is an economically important shellfish in aquaculture, yet its sex determination mechanism remains unclear. Based on the re-sequencing data, candidate sexually dimorphic loci, including 281 SNPs and 21 InDels, were screened out in small abalone. The candidate sexually dimorphic SNPs were located on thirteen chromosomes but mainly on Chr. 3 (43.4%) and Chr. 9 (26.7%); while the candidate sexually dimorphic InDels were located on six chromosomes and also mainly on Chr. 3 (38.1%) and Chr. 9 (26.7%). After being checked with Integrative Genomic Viewer (IGV) and PCR, a dominant marker (SD3) based on three consecutive sexually dimorphic SNPs on Chr. 3 and a codominant marker (SC9) based on a male-specific deletion on Chr. 9 were developed to distinguish the genetic sex in small abalone. PCR validation gained 100% consistence for both sex-specific markers in the progenies of Taiwan population and an intra-hybrid variety with male parents originated from Tokyo population. The successful development of the male-specific markers suggested that small abalone has a male heterogametic sex determination and may have two sex-linked chromosomes, and also provided important tools and clues for further studies on mechanism of sex determination in small abalone.

Molecular characterization and expression analysis of an antimicrobial peptide, mytimacin-6, in the small abalone, Haliotis diversicolor

Macin is a family of antimicrobial peptides (AMPs) and is involved in the immune responses of marine mollusks. In the present study, a novel Mytimacin (denoted as Hd-mtmc 6) was identified from the small abalone Haliotis diversicolor by RNA-seq and RACE techniques. Hd-mtmc 6 contained a coding sequence of 243 bp and encoded 80 amino acids, with a putative peptide mytimacin 6 consisting of 61 amino acid residues. The mature peptide of Hd-mtmc 6 exhibited typical characteristics of AMPs, including net positive charge (+4), higher hydrophobic residue ratio (37%), and lower molecular mass. Eight cysteines in the mature peptide formed four disulfide bond bridges (C1-C6, C2-C5, C3-C7, and C4-C8). Moreover, the presence of the macin domain, a three-dimensional structure similar to that of hydramacin-1, and a phylogenetic relationship suggested that Hd-mtmc 6 could be a new member of the invertebrate macin family. In unchallenged abalone, the Hd-mtmc 6 transcript was expressed in all tested tissues and could be detected at different stages of embryonic development. Vibrio harveyi challenge caused a significant Hd-mtmc 6 transcripts upregulation within 2 - 6 h in the mantle and hepatopancreas. Our results suggested that Hd-mtmc 6 may be involved in innate immune responses of small abalone. Further investigations are required to confirm its antibacterial activity at the protein level.

The Enhanced Immune Protection in Small Abalone Haliotis diversicolor Against a Secondary Infection With Vibrio harveyi

In recent years, more and more studies have shown that early pathogenic bacterial infection in invertebrates can enhance immunity and significantly reduce mortality when reinfected with the same pathogen. There are mechanisms to explain this phenomenon, but they are relatively few. In addition, dose-dependent primary infection is also associated with increased immunity. In the present study, the initial infection dose and mortality of abalone Haliotis diversicolor after reinfection with Vibrio harveyi were recorded, and the mechanism of immune enhancement was investigated by the transcriptomic response of abalone after two successive stimuli with V. harveyi. Priming with different concentrations of pathogen can enhance immunity; however, higher concentration is not always better. Compared with the first exposure, more genes were up-regulated after the second exposure. Among the commonly expressed genes, the immune related genes were significantly or persistently highly expressed after two infections and included pattern recognition receptors as well as immune effectors, such as toll-like receptors, perlucin 4, scavenger receptor class B-like protein, cytochrome P450 1B1-like, glutathione S-transferase 6, lysozyme and so on; in addition, these immune-related genes were mainly distributed in the pathways related to phagocytosis and calcium signaling. Among the specifically expressed genes, compared with the first infection, more genes were involved in the immune, metabolic and digestive pathways after the second infection, which would be more conducive to preventing the invasion of pathogens. This study outlined the mechanism of immune enhancement in abalone after secondary infection at the global molecular level, which is helpful for a comprehensive understanding of the mechanism of immune priming in invertebrates.

Regulatory effect of heat shock transcription factor-1 gene on heat shock proteins and its transcriptional regulation analysis in small abalone Haliotis diversicolor

BackgroundThe effects of diverse stresses ultimately alter the structures and functions of proteins. As molecular chaperones, heat shock proteins (HSPs) are a group of highly conserved proteins that help in the refolding of misfolded proteins and the elimination of irreversibly damaged proteins. They are mediated by a family of transcription factors called heat shock factors (HSFs). The small abalone Haliotis diversicolor is a species naturally distributed along the southern coast of China. In this study, the expression of HdHSF1 was inhibited by RNAi in hemocytes in order to further elucidate the regulatory roles of HdHSF1 on heat shock responsive genes in abalone. Meanwhile, to understand the transcriptional regulation of the HdHSF1 gene, the 5′-upstream regulatory region of HdHSF1 was characterized, and the relative promoter activity was examined by dual-luciferase reporter gene assay system in HEK293T cell lines.ResultsAfter the inhibition of the H. diversicolor HSF1 gene (HdHSF1) by dsRNA (double-stranded RNA), the expression of most heat shock related-genes was down-regulated (p < 0.05). It indicated the importance of HdHSF1 in the heat shock response of H. diversicolor. Meanwhile, 5′-flanking region sequence (2633 bp) of the HdHSF1 gene was cloned; it contained a putative core promoter region, TATA box, CAAT box, CpG island, and many transcription elements. In HEK293T cells, the 5′-flanking region sequence can drive expression of the enhanced green fluorescent protein (EGFP), proving its promoter function. Exposure of cells to the high-temperature (39 °C and 42 °C) resulted in the activation of HdHSF1 promoter activity, which may explain why the expression of the HdHSF1 gene participates in heat shock response. Luciferase activity of different recombinant plasmids, which contained different truncated promoter fragments of the HdHSF1 gene in HEK293T cells, revealed the possible active regions of the promoter. To further identify the binding site of the critical transcription factor in the region, an expression vector with the site-directed mutation was constructed. After being mutated on the GATA-1 binding site, we found that the luciferase activity was significantly increased, which suggested that the GATA-1 binding site has a certain weakening effect on the activity of the HdHSF1 promoter.ConclusionsThese findings suggest that GATA-1 may be one of the transcription factors of HdHSF1, and a possible signaling pathway mediated by HdHSF1 may exist in H. diversicolor to counteract the adverse effects of heat shock stress.

Physiological Responses of Intrinsic Small Abalone Haliotis diversicolor aquatilis under High Temperature Stress by Low Level 60CO Gamma Irradiation-Mediated Hormetic Effect

Haliotis diversicolor aquatilis (H. diversicolor aquatilis) is one of the aquaculture abalone species in Jeju Island, South Korea. The high water temperature in the middle of the summer season frequently limits the aquaculture productivity of abalone. To cope with the problem, this study aimed to investigate viability, attachment rate, changes of immune response, and physiological properties of juvenile small abalone Haliotis diversicolor aquatilis against high water temperature stress under 60CO-gamma irradiation-mediated hormetic effects. To examine physiological changes of abalone, the gamma-ray irradiated and non-irradiated groups were distinguished by grouping a total of 70 abalones in a treated group and a control group (each group included twenty-five female and ten male abalones). The treated group was exposed to 60CO-gamma irradiation using a designed experimental apparatus, the control group was not. Our results revealed that the low level of gamma ray (20 Gy)-irradiated Haliotis diversicolor aquatilis showed a 100% survival rate during the experiment. After gamma ray exposure, all the abalones were cultured without feeding and bioactivities were measured to examine gamma ray-induced physiological responses. The results suggested the potential for selective breeding using gamma ray irradiation hormesis to manipulate the number of eggs, fertilization rate, hatching rate, and attachment rate. The shell length of juvenile abalone was significantly enhanced by a 20 Gy radiation dose. We could presume that the effect of hormesis in the gamma-ray irradiation parent shellfish also had a genetic effect on the offspring. In order to verify changes in immune response and stress tolerance of abalone under high temperature stress, lysozyme activity and survival rates were compared at a water temperature of 30 °C. Interestingly, the 60Co gamma ray-irradiated abalones exhibited almost a 1.65-fold enhanced survival rate along with reduced lysozyme activity after 12 h of high temperature stress. Our results speculate that low levels of 60Co gamma ray-mediated hormetic effects can be an effective strategy for shell length growth and high temperature stress tolerance.

Molecular characterization and immune analysis of a defensin from small abalone, Haliotis diversicolor.

As one of antimicrobial peptides (AMPs), defensins are involved in invertebrate innate immunity against invading pathogens. In this study, a member of the invertebrate defensins was cloned and characterized from the small abalone Haliotis diversicolor, designated HdDef-2. The HdDef-2 cDNA contained a 201 bp open reading frame encoding 66 amino acids including a signal peptide of 18 amino acids and a mature peptide of 48 amino acids. The mature peptide of HdDef-2 possessed similar features to other AMPs, such as lower molecular mass, net positive charge (+1), and a high hydrophobic residue ratio (45%). In addition, six cysteines in the mature peptide were arranged in the pattern C-X16-C-X3-C-X9-C-X4-C-X1-C and stabilized the α-helix/β-sheet motif (CSαβ) with three disulfide bonds (C1-C4, C2-C5 and C3-C6) in the predicted tertiary structure. Moreover, the similar three-dimensional structure to Anopheles gambiae defensin and a phylogenetic analysis suggest that HdDef-2 may be a new member of the arthropod defensin family. Quantitative real-time PCR analysis revealed that HdDef-2 transcripts were constitutively expressed in the mantle, gill, hepatopancreas, and foot, with the highest level in the hepatopancreas. It was observed that HdDef-2 transcripts were significantly induced in the hepatopancreas after infection by Vibrio harveyi. These results indicate that HdDef-2 may be involved in the immune response against invading pathogenic bacteria, but future work is needed to verify its antimicrobial activity in protein level and elucidate the underlying mechanisms.

Integrative transcriptome analysis and discovery of genes involving in immune response of hypoxia/thermal challenges in the small abalone Haliotis diversicolor

In recent years, the abalone aquaculture industry has been threatened by the deteriorating environmental conditions, such as hypoxia and thermal stress in the hot summers. It is necessary to investigate the molecular mechanism in response to these environmental challenges, and subsequently understand the immune defense system. In this study, the transcriptome profiles by RNA-seq of hemocytes from the small abalone Haliotis diversicolor after exposure to hypoxia, thermal stress, and hypoxia plus thermal stress were established. A total of 103,703,074 clean reads were obtained and 99,774 unigenes were assembled. Of the 99,774 unigenes, 47,154 and 20,455 had homologous sequences in the Nr and Swiss-Prot protein databases, while 16,944 and 10,840 unigenes could be classified by COG or KEGG databases, respectively. RNAseq analysis revealed that the differentially expressed genes (DEGs) after challenges of hypoxia, thermal stress, or hypoxia plus thermal stress were 24,189, 29,165 and 23,665, among which more than 3000 genes involved in at least 230 pathways, including several classical immune-related pathways. The genes and pathways that were involved in immune response to hypoxia/thermal challenges were identified by transcriptome analysis and further validated by quantitative real-time PCR and RNAi technology. The findings in this study can provide information on H. diversicolor innate immunity to improve the abalone aquaculture industry, and the analysis of the potential immune-related genes in innate immunity signaling pathways and the obtained transcriptome data can provide an invaluable genetic resource for the study of the genome and functional genes.

Studies on the Development of Small Abalone (Haliotis diversicolor supertexta) Essence and its Antioxidative Properties

ABSTRACTEssences of small abalone (Haliotis diversicolor supertexta) were prepared by two methods, steaming and boiling. The chemical constituents and antioxidative properties of these essences were determined and compared to those of commercial freshwater clam essence (CFCE). Boiled small abalone essence (BSAE) contained more total soluble solids than steamed small abalone essence (SSAE). Total soluble solids and peptide content of both essences increased with longer extraction time. Glycogen contents of SSAE, BSAE, and CFCE were 14.01, 13.95, and 10.74 mg/mL, respectively. The concentrations of free amino acids in SSAE and BSAE (725.05 and 818.56 mg/100 g, respectively) were significantly higher than in CFCE (33.57 mg/100 g). The antioxidative properties of SSAE and BSAE were higher than that of CFCE. The reducing power and ferrous iron (Fe2+) chelating activity of 3-h SSAE were significantly higher than those of 10-min BSAE. The predominant free amino acid of both SSAE and BSAE was taurine (227.14–273.64 mg/100 g), followed by proline and arginine. Both essences also showed high correlations between peptide content and Fe2+ chelating activity and reducing power. Sensory evaluation trials suggested that 3-h SSAE is more favorable than BSAE in terms of appearance and overall acceptability. In conclusion, SSAE had better antioxidative properties and sensory scores than BSAE.

Proteomic analysis of trochophore and veliger larvae development in the small abalone Haliotis diversicolor

BackgroundHaliotis diversicolor is commercially important species. The trochophore and veliger are distinct larval stages in gastropod development. Their development involves complex morphological and physiological changes. We studied protein changes during the embryonic development of H. diversicolor using two dimensional electrophoresis (2-DE) and label-free methods, tandem mass spectrometry (MS/ MS), and Mascot for protein identification.ResultsA total of 150 2-DE gel spots were identified. Protein spots showed upregulation of 15 proteins and downregulation of 28 proteins as H. diversicolor developed from trochophore to veliger larvae. Trochophore and veliger larvae were compared using a label-free quantitative proteomic approach. A total of 526 proteins were identified from both samples, and 104 proteins were differentially expressed (> 1.5 fold). Compared with trochophore larvae, veliger larvae had 55 proteins upregulated and 49 proteins downregulated. These differentially expressed proteins were involved in shell formation, energy metabolism, cellular and stress response processes, protein synthesis and folding, cell cycle, and cell fate determination. Compared with the 5 protein (fructose-bisphosphate aldolase, 14–3-3ε, profilin, actin-depolymerizing factor (ADF)/cofilin) and calreticulin) expression patterns, the mRNA expression exhibited similar patterns except gene of fructose-bisphosphate aldolase.ConclusionOur results provide insight into novel aspects of protein function in shell formation, torsion, and nervous system development, and muscle system differentiation in H. diversicolor larvae. “Quality control” proteins were identified to be involved in abalone larval development.

Genetic diversity and stock identification of small abalone (Haliotis diversicolor) in Taiwan and Japan.

Small abalone (Haliotis diversicolor) is a commercially valuable species for both fisheries and aquaculture. The production of annual farmed small abalone in Taiwan, once the highest in the world, has dramatically decreased in the past 15 years, and currently, the industry is close to collapse. Understanding the genetic diversity of small abalone and developing stock identification methods will be useful for genetic breeding, restoring collapsed stocks, managing stocks, and preventing illegal trade. We investigated 307 cultured and wild individuals from Taiwan, Japan, and Bali Island (Indonesia) by using the mitochondrial cytochrome c oxidase subunit I (COI) gene. Network analysis of mtDNA COI gene sequences revealed that the individuals collected from Taiwan, Japan, and Indonesia could be identified, and showed significant genetic divergence. In addition, the Indonesian population (Haliotis diversicolor squamata) was significantly different from the other populations and might need to be considered a separate species. We discovered a single nucleotide polymorphism marker in the mtDNA COI gene that can be used to distinguish the Taiwan population from the Japan population. We also developed a polymerase chain reaction-restriction fragment length polymorphism method for rapid detection. Furthermore, we could identify the cultured stocks, wild population, and hybrid stocks by using 6 microsatellites and amplified fragment length polymorphism. This study contributes useful tools for stock identification and the production of high-disease resistant small abalone strains (Japan × Taiwan or Taiwan × Japan). Efforts should be made to avoid unintentional random genetic mixing of the Taiwan population with the Japan population and subsequent breakdown of population differentiation, which impair local adaptation of the Taiwan wild population. Molecular markers revealed a split between the Taiwan and Japan populations, and the existence of a possible barrier to the free dispersal of small abalone is discussed.

The characterization of RHEB gene and its responses to hypoxia and thermal stresses in the small abalone Haliotis diversicolor.

RHEB (Ras Homolog Enriched in Brain) is a GTP-binding protein that is ubiquitously expressed in humans and other mammals. The protein is largely involved in the mechanistic target of rapamycin (mTOR) pathway, and regulates the cell cycle progression and growth. The goal of this study was to characterize the RHEB gene in the small abalone Haliotis diversicolor, and identify the responses of RHEB gene to stresses of hypoxia or/and thermal. The objectives were to: 1) clone the full-length cDNA RHEB gene in the H. diversicolor (HdRHEB); 2) quantify the expression of HdRHEB gene in tissues of haemocytes, mantle, kidney, gill, digestive tract, colleterial gland, and hepatopancreas by using RT-PCR, and 3) evaluate the responses of HdRHEB in gill and haemocyte to stresses of hypoxia (0.2mg/l00ml), thermal (31°C), and combination of hypoxia (0.4mg/l00ml) and thermal (30°C) at exposure time of 0, 4, 24, 96, and 192h. The full length cDNA of HdRHEB was 1044bp encoding a peptide of 182 amino acid residues. Expression of HdRHEB gene was detected in all of the 7 tissues and showed the highest in mantle (P<0.05). Under hypoxia, expression of HdRHEB in gill increased significantly at 4h, 24h and 96h (P<0.05), and that in haemocyte increased significantly at 24h, 96h and 192h (P<0.05). Under thermal stress, expression of HdRHEB gene in gill decreased significantly at 4h and 24h, while expression in haemocyte decreased significantly all the time. Under thermal and hypoxia stresses, expression of HdRHEB gene in gill and haemocyte was up-regulated significantly at 24h and 96h (P<0.05). The results in this study demonstrated for the first time that RHEB gene in abalones is able to response to stress stimuli of hypoxia or/and thermal.

PI3K-AKT signaling pathway is involved in hypoxia/thermal-induced immunosuppression of small abalone Haliotis diversicolor

The PI3K-AKT signal pathway has been found to be involved in many important physiological and pathological processes of the innate immune system of vertebrates and invertebrates. In this study, the AKT (HdAKT) and PI3K (HdPI3K) gene of small abalone Haliotis diversicolor were cloned and characterized for the important status of PI3K and AKT protein in PI3K-AKT signaling pathway. The full length cDNAs of HdAKT and HdPI3K are 2126 bp and 6052 bp respectively, encoding proteins of 479 amino acids and 1097 amino acids, respectively. The mRNA expression level of fourteen genes in the PI3K-AKT signaling pathway were detected by quantitative real-time PCR. The results showed that all these fourteen genes were ubiquitously expressed in seven selected tissues. Meanwhile, HdAKT was expressed in haemocytes with the highest expression level (p < 0.05) next in hepatopancreas (p < 0.05). On the other hand, the expression level of HdPI3K in haemocytes was higher than other tissues. Under normal condition, the gene expression level of HdAKT, HdPI3K, and other PI3K-AKT signaling pathway members were significantly up-regulated by Vibrio parahaemolyticus infection which demonstrated that HdAKT, HdPI3K, and other PI3K-AKT signaling pathway members play a role in the innate immune system of abalone. The mRNA expression of these genes in gills, haemocytes and hepatopancreas was significantly down-regulated after the Vibrio parahaemolyticus stimulation with environment stimulation (thermal, hypoxia and thermal & hypoxia). These results indicate that the dual/multiple stresses defeat the immune system and lead to immunosuppression in abalone. PI3K-AKT signaling pathway may be involved in hypoxia/thermal-induced immunosuppression of small abalone Haliotis diversicolor.

Genetic mapping and quantitative trait loci analysis of growth-related traits in the small abalone Haliotis diversicolor using restriction-site-associated DNA sequencing

The small abalone (Haliotis diversicolor) is an important aquaculture species in southern China. Traditional breeding programs have been implemented for this species. But due to long reproduction cycle of the small abalone, the traditional method takes too long. There is therefore an urgent need to accelerate molecular breeding in H. diversicolor. Here we report on the construction of a high-density genetic map and quantitative trait loci detection in the small abalone using an F1 cross population with 131 progeny based on restriction-site-associated DNA sequencing. The sex-averaged map spanned 2190.1cM with a mean distance between single nucleotide polymorphisms (SNPs) of 0.59cM. A total of 3717 SNPs were mapped on 16 linkage groups. There were 1728 SNPs on the female map and 2673 SNPs on the male map. The total length of the female and male map was 2197.8 and 1764.7cM, respectively. Six growth-related traits (body weight, muscle weight, shell weight, shell length, shell width and shell height) were recorded to detect QTL. A total of 15 suggestive QTL were identified and most of them were found on LG11. A consensus QTL for six growth-related traits was detected on LG11. It suggests that these traits may be controlled by a common region. Our results would provide valuable tools which can be used for Marker-Assisted Selection (MAS) in breeding programs of H. diversicolor. Statement of relevanceThis paper published a high-density linkage map and QTL analysis for growth-related traits. The study will be valuable tools for in Marker-Assisted Selection breeding program in Haliotis diversicolor.

IGFBP7 is involved in abalone metamorphosis

Metamorphosis is the most critical life stage for abalone. There are morphogenetic transformations and habitat selection with metamorphosis. A large amount of larvae often die during this stage. Insulin/insulin-like growth factor signaling pathway (IIS) plays a role in the development. However, there is little knowledge of IIS function in metamorphosis. IGFBP7, a component of IIS, was found to be involved in metamorphosis of small abalone Haliotis diversicolor. The expression of saIGFBP7 mRNA rapidly increased before metamorphosis, which were mainly localized in the velum and foot. There was a significant decrease of metamorphosis rate after competent larvae were exposed to 5μg/mL dsRNA of saIGFBP7 (p=2.50×10−10, F=389.05). The expressions of three downstream genes of IIS (PI3K, ERKα and AKT) as well as saIGFBP7 were decreased in the dsRNA exposure experiment. There was no significant difference of metamorphosis rate (p=0.97, F=0.071) after competent larvae were induced in different concentrations of insulin. The expression of saIGFBP7, PI3K and ERKα also had no significant difference in insulin inducing assay. However, AKT expression level of 1μmol/L insulin induced group was significantly higher than that of other concentrations (p=3.41×10−7, F=26.68). These results suggested that IGFBP7 is involved in metamorphosis of abalone larvae. Our study provides insight on developing new methods for increasing the metamorphosis rate in abalone aquaculture. However, it does not work as a negative regulator of IIS activity, or plays its role in an IGF- and insulin-independent manner during metamorphosis of abalone larvae.

Construction of the BAC Library of Small Abalone (Haliotis diversicolor) for Gene Screening and Genome Characterization.

The small abalone (Haliotis diversicolor) is one of the most important aquaculture species in East Asia. To facilitate gene cloning and characterization, genome analysis, and genetic breeding of it, we constructed a large-insert bacterial artificial chromosome (BAC) library, which is an important genetic tool for advanced genetics and genomics research. The small abalone BAC library includes 92,610 clones with an average insert size of 120 Kb, equivalent to approximately 7.6× of the small abalone genome. We set up three-dimensional pools and super pools of 18,432 BAC clones for target gene screening using PCR method. To assess the approach, we screened 12 target genes in these 18,432 BAC clones and identified 16 positive BAC clones. Eight positive BAC clones were then sequenced and assembled with the next generation sequencing platform. The assembled contigs representing these 8 BAC clones spanned 928 Kb of the small abalone genome, providing the first batch of genome sequences for genome evaluation and characterization. The average GC content of small abalone genome was estimated as 40.33%. A total of 21 protein-coding genes, including 7 target genes, were annotated into the 8 BACs, which proved the feasibility of PCR screening approach with three-dimensional pools in small abalone BAC library. One hundred fifty microsatellite loci were also identified from the sequences for marker development in the future. The BAC library and clone pools provided valuable resources and tools for genetic breeding and conservation of H. diversicolor.

Involvement of Antizyme Characterized from the Small Abalone Haliotis diversicolor in Gonadal Development.

The small abalone Haliotis diversicolor is an economically important mollusk that is widely cultivated in Southern China. Gonad precocity may affect the aquaculture of small abalone. Polyamines, which are small cationic molecules essential for cellular proliferation, may affect gonadal development. Ornithine decarboxylase (ODC) and antizyme (AZ) are essential elements of a feedback circuit that regulates cellular polyamines. This paper presents the molecular cloning and characterization of AZ from small abalone. Sequence analysis showed that the cDNA sequence of H. diversicolor AZ (HdiODCAZ) consisted of two overlapping open reading frames (ORFs) and conformed to the +1 frameshift property of the frame. Thin Layer chromatography (TLC) analysis suggested that the expressed protein encoded by +1 ORF2 was the functional AZ that targets ODC to 26S proteasome degradation. The result demonstrated that the expression level of AZ was higher than that of ODC in the ovary of small abalone. In addition, the expression profiles of ODC and AZ at the different development stages of the ovary indicated that these two genes might be involved in the gonadal development of small abalone.

IGFBP7 promotes hemocyte proliferation in small abalone Haliotis diversicolor, proved by dsRNA and cap mRNA exposure

Insulin-like growth factor binding protein 7 (IGFBP7) binds IGFs with a low affinity, but in contrast, recognizes insulin with a high affinity. Many studies show that IGFBP7 involves several cellular processes of vertebrates and functions as a tumor suppressor gene in different tumors. However, the function of IGFBP7 in invertebrates is unclear. In this research, we studied the function of IGFBP7 in the proliferation of small abalone Haliotis diversicolor hemocytes by exposure to dsRNA or cap mRNA of saIGFBP7. We found that exposure to dsRNA or cap mRNA of saIGFBP7 could significantly affect the mRNA and protein expression of IGFBP7 in cultured small abalone hemocytes (p<0.05). There was a significant increase in hemocyte density and the number of adherent hemocytes after exposure to cap mRNA of saIGFBP7 (p<0.05). Similarly, exposure to dsRNA of saIGFBP7 could significantly decrease the hemocyte density and the number of adherent hemocytes (p<0.05). These findings suggest that IGFBP7 increases hemocyte growth. It is the first time to report the effect of IGFBP7 on the proliferation of marine invertebrate cells.

Molecular cloning, expression and functional analysis of a predicted HdiQM gene in small abalone,Haliotis diversicolor

The small abalone (Haliotis diversicolor) is a mollusc and was cultured in south of china. Problems such as the decreasing pathogen-resistance and their mass mortality during the summer. The increased immunity of small abalone populations is a key factor in resolving these problems. Thus, the study of immunity-related genes in small abalone has become important. In this study, three bacterial species were initially isolated from small abalone carcasses. The regression of infection was analysed, which revealed that the bacteria species could cause rapid morbidity in small abalone. A QM-like gene (HdiQM) was found and bacterial challenge tests showed that HdiQM gene expression was induced by the bacterial isolates from small abalone carcasses. Therefore, our results implied that HdiQM was found to be an inflammatory stress-inducible gene associated with pathogen infection, with important functions in small abalone immunity.

Genetic parameters and selection responses for growth and survival of the small abalone Haliotis diversicolor after four generations of successive selection

Abstract Genetic parameters and selection responses were estimated for growth and survival of the small abalone Haliotis diversicolor using a fully pedigreed synthetic population from two Chinese hatchery stocks and two wild stocks. About 53,300 progeny representing 533 full-sib families from the mating between 308 sires and 533 dams were tested in concrete tanks for five generations. Breeding candidates in the base generation (G 0 ) and first generation (G 1 ) were selected based on their estimated breeding values for growth (recorded as shell length at tagging and harvest), while those in later generations (G 2 –G 3 ) were selected according to a selection index including individual breeding values for growth and family breeding values for survival traits. Variance components and genetic parameters were estimated using animal models and the restricted maximum-likelihood method. The breeding values of all animals over five generations were estimated using the best linear unbiased prediction. Within generations, the heritability estimates for square root transformed tagging shell length and harvest shell length were moderate, ranging from 0.125 to 0.237 and from 0.196 to 0.308, respectively. Across generations, the heritability estimates for the two growth traits were relatively low; 0.109 and 0.169, respectively, and the heritability of survival was low (0.069), but suggestive of a significant additive genetic component. Including all data, the effects common to full-sibs other than additive genetic effects ( c 2 ) accounted for 5.1%, 2.6% and 3.1% of the total phenotypic variance for transformed tagging shell length, harvest shell length and survival traits, respectively, and the three values were significantly different from zero (P H. diversicolor.