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Prostaglandin E2 (PGE2) has been reported to be involved in reproduction as one of the most abundant and widely distributed prostaglandins in invertebrates. Regulating gonad development and gamete production in mollusks can greatly assist artificial breeding, which is believed to promote the commercial benefits of various mollusk species. In the present study, the contents of PGE2 in the gonads of abalone at different developmental stages (maturing stage, ripe stage, and spawning stage) and reproduction states (before and after spawning) were measured. A higher PGE2 level was found in the gonads of abalones at the ripe stage than other stages, and the highest PGE2 level was detected in the ovaries of female H. diversicolor in the UV-stimulated group. Then the effects of PGE2 injection on the spawning efficiency were explored, and the gamete emission rates of female and male abalone were significantly increased after PGE2 injection. Through qRT-PCR and in situ hybridization assay, higher expression levels and wide distribution of phospholipase A2 (PLA2), cyclooxygenase 2 (COX2), prostaglandin E synthases (PGES2), and prostaglandin E2 receptor 4 (EP4) in the gonads of H. diversicolor were observed after spawning. It is speculated that PGE2 may promote the reproductive cycle in both male and female H. diversicolor, although the regulation mechanism of PGE2 in female and male abalone might be different. The results will be useful for the application of PGE2 in the abalone aquaculture practices to increase the reproduction efficiency in future. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs), a member of pattern recognition receptors, play an important role in the innate immunity by recognizing invading microorganisms. In this study, a novel perlucin gene (designated as HdPer 3), a typical CTLs was cloned and characterized from the small abalone Haliotis diversicolor. The open reading frame of HdPer 3 was 471 bp, encoding a protein of 156 amino acids that included a single carbohydrate-recognition domain. HdPer 3 was widely expressed in all tested tissues and developmental stage. HdPer 3 expression was significantly up-regulated after Vibrio harveyi infection, suggesting that HdPer 3 was activated in response to bacterial infection. The encapsulation ability of hemocytes could be significantly enhanced by the recombinant protein HdPer 3 (rHdPer 3). To understand the regulation mechanism of the HdPer 3 , HdPer 3 was silenced in vivo by RNAi. Knocking down HdPer 3 decreased the hemocytes phagocytosis. Meanwhile, knocking down HdPer 3 can reduce the expression of 2 phagocytosis-related genes (Rab and Dynamin), TNF-α, and 2 MAPK pathway-related genes (MAPK-X1 and Ras) after V. harveyi infection. Moreover, HdPer 3 interference could increase the bacterial load in the hemolymph and the mortality of abalones after V. harveyi infection. All these results suggested that HdPer 3 played a crucial role in the defense against V. harveyi infection by recognizing bacterial pathogens and activating the expression of immune-related genes. • Identified a novel C-type lectin, perlucin, from the small abalone. • Knocking down HdPer 3 decreased phagocytosis. • Knocking down HdPer 3 increased the mortality of the small abalone after V. harveyi infection. [ABSTRACT FROM AUTHOR]

Bacteria affiliated to the Deltaproteobacteria dominate intestinal microbial communities in the abalone, Haliotis diversicolor, and may colonize the host's digestive gland, raising the possibility that they contribute to digestion of macroalgae. However, the phylogenetic, and genomic and metabolic characteristics of these Deltaproteobacteria, and the nature of their relationship to abalone host remain elusive. Here, we examined the intestinal microbial composition of H. diversicolor using high‐throughput sequencing and described the genomic characteristics of the Deltaproteobacteria phylotype using genome‐centric metagenomics. High‐throughput sequencing confirmed that one Deltaproteobacteria phylotype was predominant in intestinal microbiota of H. diversicolor. Phylogeny analysis based on full‐length 16S rRNA gene allocated this bacterium to a distinct and unique cluster within SAR324. It possessed a genome of at least 1.59 Mb with 35.15 mol% GC content, much smaller than other sequenced free‐living SAR324 bacteria. According to genome annotation and metabolic reconstruction, ATP binding cassette transporters for sugars and carbohydrate metabolism pathways indicated heterotrophic potential. Interestingly, it encoded polysaccharide lyases, which, if expressed, could help the host to digest its macroalgae diet. In contrast to the known SAR324, no sulphur oxidation or carbon fixation pathways were predicted. We propose that this is a unique and specific SAR324 bacterium in symbiosis with Haliotis. [ABSTRACT FROM AUTHOR]

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 Hdmtmc 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 threedimensional 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. [ABSTRACT FROM AUTHOR]

Due to increased temperatures and aquaculture density, thermal and hypoxia stresses have become serious problems for the aquaculture of abalone Haliotis diversicolor. Stresses lead to immunosuppression, which can cause severe negative impacts on aquaculture farms. To study the mechanism of immunosuppression after hypoxia stress and bacterial challenge, transcriptomes of H. diversicolor hemocytes involved in immunity were profiled. A total of 307,395,572 clean reads were generated and assembled into 99,774 unigenes. KEGG analysis indicated that 225 unigenes with immunologic function were mapped into immune-related pathways. Expression of 41 unigenes measured by quantitative real-time PCR (qRT-PCR) showed consistent results with that of transcriptome analysis. When exposure challenge of Vibrio parahaemolyticus, it is indicated that the PI3K-AKT, MAPK, NF-κB and P53 signal pathways were involved in the hypoxia-induced immunosuppression of H. diversicolor. Furthermore, when the AKT gene (HdAKT) was inhibited by double-stranded RNA (dsRNA), expression levels of HdAKT was lower than the blank and control group in hemocytes at 4 h, 12 h and 24 h (p < 0.05). [ABSTRACT FROM AUTHOR]

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-X 16 -C-X 3 -C-X 9 -C-X 4 -C-X 1 -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. Unlabelled Image • Haliotis diversicolor defensin-2 (denoted as HdDef-2) shares the common features of antimicrobial peptides. • HdDef-2belongs to the arthropod defensins. • The main tissue expressing HdDef-2 was found to be the hepatopancreas. • HdDef-2 was significantly induced following Vibrio harveyi infection. [ABSTRACT FROM AUTHOR]

The variously colored abalone Haliotis diversicolor is widely distributed in the northwest Pacific region and commonly occurs in shallow subtidal regions of Jeju Island, off the south coast of Korea. In this study, we first characterized the morphology and immune-related activities of H. diversicolor hemocytes using flow cytometry and light microscopy. Flow cytometry revealed three types of hemocytes in the hemolymph: granulocytes, hyalinocytes, and blast-like cells. Hyalinocytes were the largest and most abundant, while granulocytes were intermediate-sized and contained many granules in the cytoplasm. Flow cytometry indicated that granulocytes were mainly involved in phagocytosis and oxidative activities, although hyalinocytes also exhibited a certain level of immune-related activities. Blast-like cells were the smallest and exhibited a lack of phagocytosis and oxidative capacities. To understand the adaptive capacity to air exposure, abalones were exposed to air or humidity over 30 h. All abalones perished within 24 h after dry-air exposure, while 20% of abalones had survived under humid conditions by the end of the experiment. After 6 h of air exposure and 12 h of humidity, total hemocyte counts and phagocytosis capacity declined dramatically, and the level of hemocyte DNA damage increased significantly (P <.05). This study suggests that sufficient oxygen supply during transportation could enhance the survival of H. diversicolor , thereby improving its market value. • We first characterized types of hemocyte in the abalone Haliotis diversicolor using flow cytometry. • Granulocyte, hyalinocyte, and blast-cell like cell were identified, while the granulocyte were mainly involved in immune response. • After 6 h of air exposure, the immune capacities of the abalone declined dramatically and DNA damage increased significantly. [ABSTRACT FROM AUTHOR]

Abstract Hemocyanins (Hc), the main protein components of hemolymph in invertebrates, are not only involved in oxygen transport but also linked to non-specific immune responses. In this study, we used abalone (Haliotis diversicolor) Hc to study the basis of its diversified functions through gene, protein, peptides, and phenoloxidase (PO) activity levels. Three complete hemocyanin gene (HdH) sequences were cloned for the first time. By comparing the copies and location of HdH between abalone and other mollusks, we propose that Hc gene duplication and linkage is likely to be common during the evolution of mollusk respiratory proteins. We further demonstrate that all three genes could be expressed in abalone, with expression varying based on the developmental stages, tissue types, and different pathogen infections. However, HdH1 and HdH2 appear to be synthesized by the same cells by fluorescence in situ hybridization. Furthermore, the PO activity of HdH can be induced by trypsin, urea, and SDS in vitro. Viral infection can stimulate its PO activity in vivo by cleaving the protein into fragments. Consequently, we present a comprehensive study of abalone hemocyanin, providing important evidence for an in-depth understanding of the physiological and immune functions of Hc in mollusks. [ABSTRACT FROM AUTHOR]

Heavy metals in soil, water, and industrial production can affect the antibiotic resistance of bacteria. Antibiotic resistance in gut microbiota has been extensively researched. The effects of cadmium (Cd) was investigated on the gut microbiota and antibiotic resistance genes (ARGs) of Haliotis diversicolor , a commercially important abalone species. By exposing H. diversicolor to four concentrations of Cd (0 μg L−1 (control), 6.5 μg L−1 (low), 42.25 μg L−1 (medium), and 274.63 μg L−1 (high)) for 30 and 60 days, 16 types of ARG (aadA-01 , aadA-02 , cfr , dfrA1 , ermB , floR , folA , mecA , sul2 , tetB-01 , tetC-01 , tetD-01 , tetG-01 , tetM-02 , tetQ , vanC-01), and 1213 genus and 27 phylum microbiomes were detected. ARGs can be resistant to aminoglycoside, beta-lactamase, macrolide-lincosamide-streptogramin B, multidrug, florfenicol, macrolide, sulfonamides, tetracyclines, and vancomycin. Cadmium exposure significantly alters the abundance of tetC-01 , tetB-01 , tetQ , sul2 , and aadA-01. About 5% (61) of genus-level microorganisms were significantly affected by Cd exposure. Microbiota alpha and beta diversities in the 60-day 42.25 μg L−1 Cd treatment differed significantly from those in other treatments. In addition, 26 pathogens were detected, and two pathogens (Vibrio and Legionella) were significantly affected by Cd exposure. Significant correlations between pathogens and ARGs increased with increased Cd concentration after 60 days of Cd exposure. Cadmium exposure may cause gut microbiota disturbance in H. diversicolor and increase the likelihood of ARG transfer to pathogens, increasing potential ecological and economic risks. [Display omitted] • Cadmium exposure significantly altered the intestinal microbiota distribution. • Cadmium exposure alter the abundance of tetC-01, tetB-01, tetQ, sul2 and aadA-01. • Cadmium exposure was the driving factors for the variation of ARGs abundance. • Correlations between pathogens and ARGs increased with Cd concentration. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Haliotis diversicolor supertexta was highly susceptible to Haliotid herpesvirus 1 (HaHV-1-CN2003). • Haliotis discus hannai was not susceptible to HaHV-1-CN2003. • Tropism of HaHV-1-CN2003 included both neural tissue and haemocytes. Abstract Abalone viral ganglioneuritis (AVG), caused by Haliotid herpesvirus -1 (HaHV-1) infection, has been reported as the main cause of mortality and heavy losses of wild and cultivated abalone in Taiwan and Australia since 2003. HaHV-1 DNA has also been reported in diseased abalone collected in early 2000s in China. However, no data is available about the susceptibility, disease process and pathological changes of HaHV-1 infection in the primary cultivated abalone species in China. In the present study, two cultivated abalone species, Haliotis diversicolor supertexta and Haliotis discus hannai , were challenged with HaHV-1-CN2003 collected in 2003 in China using three different methods. Results showed that H. diversicolor supertexta was highly susceptible to HaHV-1-CN2003 infection and suffered acute mortality using all three challenge methods. H. discus hannai was not susceptible to the viral infection. Histopathology combined with transmission electron microscopy and quantitative PCR analysis revealed that the tropism of HaHV-1-CN2003 includes both neural tissue and haemocytes. [ABSTRACT FROM AUTHOR]

There is growing interest in the use of probiotics as an alternative strategy for the promotion of growth rates and prevention of diseases in aquaculture. In this study, abalone Haliotis diversicolor were fed with different probiotic-supplemented diets for 180 days, containing Bacillus stratosphericus A3440, Phaeobacter daeponensis AP1220, and a mixture of the two strains. Probiotic supplementation significantly increased shell length and wet weight of juvenile abalone (p < 0.05). Compared with those non-treatment group, trypsin and lipase activities were increased significantly in the gastrointestinal tract of animals fed with A3440 or bacterial mixture (p < 0.05). A subsequent ten-day of challenging trial against Vibrio harveyi , the animals with AP1220 had ~90% survival rate as against ~60% survival rate for the non-treated animals. Likewise, in situ glutathione peroxidase, catalase, alkaline phosphatase, acid phosphatase, and superoxide dismutase assays showed that the probiotics significantly improved immune response of H. diversicolor (p < 0.05). 13,622 OTUs were obtained from abalone intestine microbiome by 16S rRNA high-throughput sequencing. Microbial diversity indices and richness estimates in the non-treated group declined before and after Vibrio infection trial. Conversely, these indexes enhanced to different levels in probiotics treatments, and the A3440 group restored and reached the highest value. Cluster analysis showed that probiotic could re-establish and subsequently balance the gut microbial community after the challenge trial. Furthermore, these potential probiotics had a positive effect on the number of beneficial endogenous bacteria in the abalone gut, such as bacilli and actinobacterial species. Thus, dietary administration of B. stratosphericus , P. daeponensis and their mixture could improve the nutrient and health status of abalone H. diversicolor . [ABSTRACT FROM AUTHOR]

Abstract: Abalone (Haliotis diversicolor) is an important marine and mariculture food resource that has been severely affected by several viral disease outbreaks in recent years, and the virus causing the disease has not been unequivocally identified. To establish viral metagenomic research protocols for H. diversicolor, we applied differential centrifugation and sucrose gradient ultracentrifugation to obtain virus‐like particles from crude tissue extracts. After nuclease treatment to remove host nucleic acid, reverse transcription, and double‐stranded amplification, we used the HiSeq 2500 platform to generate a viral genome dataset. A total of 6,055,591,750 nucleotides were produced from 24,222,367 reads, generating 65,382 contigs, with a total sequence length of 14,239,389 bp. We analysed and evaluated the sequence data by quality control mapping, among other methods and the results indicated good quality data. Alignment of the data with sequences in the GenBank virus database identified homology with a large number of Abalone Herpes‐like Virus (AbHV) and Shriveling Syndrome‐associated Virus (AbSV) sequences, and also a certain amount of White Spot Syndrome Virus (WSSV) sequences. [ABSTRACT FROM AUTHOR]

Essences 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. [ABSTRACT FROM AUTHOR]

This study was conducted to determine the influence of salinity on the growth of abalone Haliotis diversicolor Reeve, including the density and size of mucous cells. Abalone individuals were reared in the laboratory at salinities of 20, 25, 31, 35 and 40 ppt. The mucous cells of the lips, gills and digestive gut of H. diversicolor, which react to some forms of stress such as suboptimal salinity, were characterized following staining with Alcian Blue-Periodic Acid-Schiff`s Reagent (AB-PAS). The specific growth rate in wet weight and shell length of H. diversicolor were highest at 31 ppt and lowest at 20 ppt (0.52 vs 0.15% d−1, and 0.058 vs 0.021 mm d−1, respectively). The abalone H. diversicolor tolerated salinity fluctuations within the range of 20-40 ppt, but growth was optimum at 25-35 ppt. Mucous cells of the lips and gills showed significant differences (ANOVA, df = 4, P = <0.001) in cell density and cell size, being less dense and larger at 31 ppt than at 40 ppt, which could be an effect of osmotic and ionic regulation. Consistent with reports in literature, salinity ranges of 25-35 ppt are suitable for growth of H. diversicolor. Results of this study indicated that areas with such salinity are favorable for stock enhancement and mariculture of the abalone H. diversicolor. [ABSTRACT FROM AUTHOR]

Background: Haliotis 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. Results: A 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 (fructosebisphosphate aldolase, 14-3-3e, profilin, actin-depolymerizing factor (ADF)/cofilin) and calreticulin) expression patterns, the mRNA expression exhibited similar patterns except gene of fructose-bisphosphate aldolase. Conclusion: Our 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. [ABSTRACT FROM AUTHOR]

RHEB ( R as H omolog E nriched in B rain) 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 (0.2 mg/l00 ml), thermal (31 °C), and combination of hypoxia (0.4 mg/l00 ml) and thermal (30 °C) at exposure time of 0, 4, 24, 96, and 192 h. The full length cDNA of HdRHEB was 1044 bp 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 4 h, 24 h and 96 h ( P < 0.05), and that in haemocyte increased significantly at 24 h, 96 h and 192 h ( P < 0.05). Under thermal stress, expression of HdRHEB gene in gill decreased significantly at 4 h and 24 h, 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 24 h and 96 h ( 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Organotin compounds, especially tributyltin (TBT) and triphenyltin (TPT), are a group of hazardous pollutants in marine environments. Haliotis diversicolor is an important marine model organism for environmental science. In this study, 1 H NMR spectroscopy together with pattern recognition methods was used to investigate the responses of hepatopancreas and gill of Haliotis diversicolor to TBT and TPT exposure. It was found that obvious gender-, tissue- and compound-specific metabolomic alterations were induced after a 28-day exposure. TBT and TPT exposure not only caused the disturbance in energy metabolism and osmotic balance in hepatopancreas and gill tissues with different mechanisms, but also induced oxidative stresses. These metabolic alterations were highlighted in the accumulation of aspartate, uridine diphosphate-N-acetylglucosamine, uridine diphosphate glucose, guanosine and the depletion of leucine, isoleucine, valine, malonate, homarine, trigonelline in all exposure gills, as well as in the depletion of ATP, AMP, betaine in male exposure gills and pantothenate in male exposure hepatopancreases. The significant decreased aromatic amino acids (AAAs), lysine and glutamate in gills and increased betaine in hepatopancreases for TPT exposure together with increased glutamate and decreased betaine in gills and increased glutamate and glycine in hepatopancreases for TBT exposure demonstrated their specific metabolic characteristics. Among these characteristic metabolites, AAAs, lysine and glutamate in the gill as well as pantothenate in the hepatopancreas might be identified as potential biomarkers for TPT or TBT exposure in Haliotis diversicolor . The results provide a useful insight into the toxicological mechanisms of organotin compounds on Haliotis diversicolor . [ABSTRACT FROM AUTHOR]

Vibrio parahemolyticus is a devastating bacterial pathogen that often causes outbreak of vibriosis in abalone Haliotis diversicolor. Elucidation of metabolic mechanisms of abalones in responding to V . parahemolyticus infection is essential for controlling the epidemic. In this work, 1 H NMR-based metabolomic techniques along with correlation and network analyses are used to investigate characteristic metabolites, as well as corresponding disturbed pathways in hepatopancreas and gill of H . diversicolor after V . parahemolyticus infection for 48 h. Results indicate that obvious gender- and tissue-specific metabolic responses are induced. Metabolic responses in female abalones are more clearly observed than those in males, which are primarily manifested in the accumulation of branched-chain amino acids and the depletion of organic osmolytes (homarine, betaine and taurine) in the infected gills of female abalones, as well as in the depletion of glutamate, branched-chain and aromatic amino acids in the infected hepatopancreases of female abalones. Moreover, based on major metabolic functions of the characteristic metabolites, we have found that V . parahemolyticus infection not only cause the disturbance in energy metabolism, nucleotide metabolism and osmotic balance, but also induce oxidative stress, immune stress and neurotoxic effect in different tissues with various mechanisms. Our study provides details of metabolic responses of abalones to V . parahemolyticus infection and will shed light on biochemical defence mechanisms of male and female hosts against pathogen infection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 ( Hdi QM) was found and bacterial challenge tests showed that Hdi QM gene expression was induced by the bacterial isolates from small abalone carcasses. Therefore, our results implied that Hdi QM was found to be an inflammatory stress-inducible gene associated with pathogen infection, with important functions in small abalone immunity. [ABSTRACT FROM AUTHOR]

This research aims to investigate the non-specific immune response of Taiwan abalone ( Haliotis diversicolor supertexta ) which was treated with the beta-1,3-1,6-glucan to be observed in the survival impact after the Vibrio alginolyticus infection. The non-specific immune and physiological response of superoxide anion radical (O 2 − ), phenoloxidase (PO), phagocytic index (PI), phagocytic rate (PR) and lucigenin-chemiluminescence for reactive oxygen intermediates (ROIs) were enhanced via in-vitro experiment. In the in-vivo experiment, the observed data presented that the haemolymph lysate supernatant (HLS), superoxide dismutase (SOD), glutamate oxalacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) were not significant enhanced, but the total haemocyte count (THC), O 2 − , PO, phagocytic index (PI), phagocytic ratio (PR) and other parameters of immune were significantly promoted after treated with beta-1,3-1,6-glucan. In the challenge experiment, the survival rates of abalone in the 40 and 80 μl/ml groups of beta-1,3-1,6-glucan were observed from 6.67% up to 33.33% and 36.67% after injection with Vibrio alginolyticus , respectively. [ABSTRACT FROM AUTHOR]

Accumulating data has demonstrated that ferritin plays an important role in host defense responses against infection by pathogens in many organisms. In this study, ultracentrifugation was used to isolate ferritin from abalone, Haliotis diversicolor , and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that this ferritin consisted of two subunits (designated as HdFer1 and HdFer2). There are no disulfide bonds between the HdFer1 and HdFer2 subunits; however, these subunits co-assemble to form heteropolymers. A novel ferritin subunit (HdFer2) was cloned from H. diversicolor by 5ʹ and 3ʹ RACE (rapid amplification of cDNA ends) approach. The full-length HdFer2 cDNA sequence consists of 878 bp with an open reading frame of 513 bp that encodes a protein that is 170 amino acids in length. Quantitative real-time PCR analysis revealed that HdFer1 and HdFer2 were transcribed in various tissues, such as the mantle, gill and hepatopancreas, with the highest levels of expression in the hepatopancreas. Following a challenge with the pathogen, Vibrio harveyi , the expression of HdFer1 and HdFer2 were markedly induced at different times. This study has identified a novel ferritin subunit in H. diversicolor which will contribute to further exploration of the role of ferritin in mollusk innate immune defense against invading pathogens. [ABSTRACT FROM AUTHOR]

Using two-dimensional gel electrophoresis, the foot muscle proteome of three geographical populations of Haliotis diversicolor were examined, with a total of 922 ± 21 protein spots detected in the Japanese population ( JJ), 904 ± 25.6 in the Taiwanese population ( TT), and 936 ± 16.2 in the Vietnamese population ( VV). Of these, 254 spots showed differential expression and 85 protein spots percentage volumes varied more than twofold. Both 'genotype' and 'spot' analysis of variance approaches significantly showed differences among the three populations. Hierarchical clustering analysis showed that TT and VV clustered together followed by clustering with JJ, which is consistent with their geographical location. Following matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, 30 differentially expressed proteins involved in major biological processes including energy production and storage and stress response were identified. Of these proteins, proteins pertaining to muscle contraction and muscle protein regulation showed highest expression levels in VV samples. Proteins involved in energy production and storage, including ATP synthase beta subunit, fructose-1,6-bisphosphate aldolase, arginine kinase, enolase, triosephosphate isomerase, and tauropine dehydrogenase, showed diverse expression patterns among the three populations. For stress-responsive proteins, the expression of heat shock protein 70 was JJ > VV > TT. The expression pattern of Cu/Zn-superoxide dismutase was JJ > VV > TT. Overall, these results may aid in the detection of new differentially expressed proteins within three different abalone populations. [ABSTRACT FROM AUTHOR]

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 2190.1 cM with a mean distance between single nucleotide polymorphisms (SNPs) of 0.59 cM. 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 1764.7 cM, 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 relevance This 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 . [ABSTRACT FROM AUTHOR]

Haliotis diversicolor is a commercially important cultured shellfish. It is also an important marine model organism for environmental science. High temperature accompanied with hypoxia frequently induces diseases or even death to abalones. In present study, 1 H NMR spectroscopy together with pattern recognition methods was used to investigate the responses of muscle and gill of H. diversicolor to thermal and hypoxic stresses. It was found that obvious gender-, time- and tissue-specific metabolic responses were induced by thermal and hypoxic stresses. In combination with the changes of H. diversicolor in physiological features, the dual-modal stresses were suggested to mainly cause the disturbance in energy metabolism and osmotic balance in muscle and gill tissues with different mechanisms. Further, the corresponding correlation networks and metabolic pathways derived from the characteristic metabolites were used to assess the major metabolic functions of these characteristic metabolites. These findings shed some lights on the metabolic influences of environmental stresses on marine organisms. [ABSTRACT FROM AUTHOR]

Abalone withering syndrome is a serious chronic disease. Ultrastructure and pathological changes were studied using transmission electron microscopy. Myofibers appeared hollow, and the number of inner intact myofibrils was reduced greatly. The morphology of mitochondria in cells appeared abnormal. Crystal lattice-like inclusions in pathological muscle cells were observed. The hepatopancreas was damaged severely; it was full of empty vesicles and devoid of any recognizable cellular structures. The activities of 3 enzymes, acid phosphatase (ACP), alkaline phosphatase (AKP), and total superoxide dismutase (T-SOD), in hemolymph of healthy and diseased abalone showed little differences. ACP activity in the pedal mucus of healthy and diseased abalone was not significantly different. However, in diseased abalone pedal mucus, activities of AKP and T-SOD were significantly lower than in the control group. In pedal mucus of healthy and diseased abalone, the differential (SDS-PAGE) bands were identified as actin and hemocyanin. Protein identification was accomplished with mass spectrometry. A total of 16 2-DE gel spots were identified; 5 gel spots showed upregulation and 11 gel spots showed downregulation in diseased abalone. Proteins involved in energy production and storage, including fructose-1, 6-bisphosphate aldolase, arginine kinase, and triosephosphate isomerase, showed diverse expression patterns in diseased abalone. For stress-responsive proteins, expression of Cu/Zn-superoxide dismutase showed downregulation. For contraction and regulation proteins of muscle, actin showed significant downregulation. Statement of relevance Abalone withering syndrome (WS) is a serious chronic disease. However, there is limited information on the physiological performance of infected abalones. The present study was to assess the alterations of Haliotis diversicolor caused by WS using transmission electron microscopy as well as assess the immune enzyme activity of hemolymph and mucus, and muscle, mucus proteins changes by 2-DE and SDS-PAGE. [ABSTRACT FROM AUTHOR]

The temperate-water Pacific abalone, Haliotis discus hannai , has replaced the local subtropical species Haliotis diversicolor , as the main cultured abalone species in southern China in recent years. Although the ability to tolerate high temperature has increased for Pacific abalone, summer mortality still has a serious effect on cultivation success, as the temperature in southern China is significantly higher than the temperature in natural habitats. Hybridization has been demonstrated as an effective way to improve adaptability to particular environmental conditions. In this study, we introduced green abalone, Haliotis fulgens , from North America into China and successfully conducted hybridization trials with Pacific abalone. Fertilization rate for H. discus hannai ♀ × H. fulgens ♂ was at an acceptable level for commercial production. The hybrids exhibited hybrid vigor for growth and survival, as well as a higher temperature tolerance, showing great potential for application in the abalone aquaculture industry in southern China. Moreover, two-step PCR methods using three primers were developed, which can be used for quick genetic identification of green abalone, Pacific abalone and their reciprocal hybrids. Statement of relevance Summer mortality still has a serious effect on cultivation success of abalone farming in China. In this study, green abalone, H. fulgens were introduced and successfully conducted hybridization trials with Pacific abalone. The hybrids exhibited exciting hybrid vigor and adaptation to the higher temperature tolerance, which showed great potentials on application to the abalone aquaculture industry in southern China. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Dermatopontin (DPT), a component of the extracellular matrix, plays important roles in cell-matrix interactions and matrix assembly. Some studies have revealed that it has more general functions in biological activities. However, its function in molluscs is poorly understood. In this study, a molluscan DPT gene, saDPT2, was cloned from small abalone Haliotis diversicolor. The full-length cDNA of saDPT2 sequence is 620 bp, with a 531 bp open reading frame encoding a protein of 177 amino acids (aa). Amino acid sequence analysis revealed that saDPT2 shares conserved signature motifs with other DPT proteins, including three repeats of 10-residue motif (S-X-H-X-N-X-Y-E-D-R), which is similar to mammalian 6-residue repeating sequence of D-R-E/Q-W-X-F/Y. Quantitative real-time PCR was employed to investigate the tissue distribution of saDPT2 mRNA, its expression at different developmental stages, and in abalone under bacteria challenge. The saDPT2 mRNA could be detected in all examined tissues and developmental stages. Moreover, the saDPT2 mRNA was up-regulated in haemocytes and gills after bacteria injection. The results indicate that the saDPT2 could respond to pathogenic infection and may play a role in adult abalone immune system. [ABSTRACT FROM AUTHOR]

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 < 0.05). Genetic correlations of harvest shell length with tagging shell length and survival were favorable and significantly different from zero; 0.633 and 0.467, respectively. The predicted response was obtained from the difference in the mean breeding values between generations. When using the LS mean of the base population (G0) as a baseline for comparison, the average selection response of harvest shell length expressed as proportions ranged from 6.1% to 12.6%, with an average of 9.2%. The average estimated selection response per population for survival was 4.1%, and the accumulated selection response was 16.5% after performing four generations of selection. Based on these results, the breeding population had considerable additive genetic variation in growth traits, and the ongoing selective breeding program resulted in considerable genetic improvement in growth and survival in H. diversicolor. [ABSTRACT FROM AUTHOR]

Inhibitor of NF-κB (IκB), nuclear factor-κB (NF-κB), and Akirin2 are all important members of Rel/NF-κB signaling pathway, which plays a pivotal role in regulating the innate immune response of vertebrates and invertebrates. In this study, the IκB ( SaIκB ) and Akirin2 ( SaAkirin2 ) cDNAs of small abalone Haliotis diversicolor were cloned and characterized. The full length cDNA of SaIκB and SaAkirin2 were 1748 bp and 1452 bp respectively, encoding a protein of 401 aa and 187 aa respectively. A conserved degradation motif (DS 56 GIYS 60 ) and six ankyrin repeats were identified in the SaIκB by SMART analysis. Meanwhile, a typical nuclear localization signal (NLS) was found at the N-terminal region of the SaAkirin2 protein. Also, the mRNA expression level of SaIκB , SaAkirin2 , and AbNF - κB were detected by quantitative real-time PCR. The results revealed that all these three genes were ubiquitously expressed in 7 selected tissues. The expression level of SaIκB in gills was higher than that in other tissues ( P < 0.05) while the expression level of AbNF - κB was significantly higher in hepatopancreas and haemocytes. The highest expression level of SaAkirin2 was detected in hepatopancreas, followed by mantle. The mRNA expression levels in either gills or haemocytes of SaIκB , SaAkirin2 , and AbNF - κB were significantly up-regulated ( P < 0.05) post thermal stress, hypoxia exposure, thermal plus hypoxia stress and the injection of Vibrio parahaemolyticus . These results indicated that these three NF-κB signaling pathway-related genes are involved in response to bacterial infection and play essential roles in response to thermal and hypoxia stress. [ABSTRACT FROM AUTHOR]

Abstract: In this study, molecular characterization and expression of three heat shock responsive genes were analyzed as indicators to understand the mechanism of heat shock response of small abalone Haliotis diversicolor under stresses. The full length cDNA of heat shock transcriptional factor 1 (HdHSF1), heat shock factor binding protein 1(HSBP1), and heat shock protein 90 (HdHSP90) are 1548 bp, 809 bp, and 2592 bp respectively, encoding a protein of 515 aa, 75 aa, and 728 aa respectively. Real time quantitative PCR analysis revealed that these three genes are constitutively expressed in 7 selected tissues. The expression level of HdHSF1 in gills was higher than that in other tissues (p < 0.05). The highest expression level of HdHSBP1 was detected in hemocytes. The highest expression level of HdHSP90 was in the digestive tract and colleterial gland. The HdHSF1 expression level in the gills was up-regulated significantly (p < 0.05) after thermal stress and hypoxia exposure respectively. On the contrary, HdHSBP1 was down-regulated both in gills and hemocytes after thermal stress and the same as in gills after hypoxia stress. HdHSP90 expression level was also up-regulated in gills and hemocytes after both thermal and hypoxia stresses. These results indicated that these three heat shock responsive genes play important roles in response to thermal and hypoxia stress. [Copyright &y& Elsevier]

In this study, hypoxia inducible factor-1α (HIF-1α) and hypoxia inducible factor-1β (HIF-1β) from small abalone Haliotis diversicolor were cloned. The cDNA of H. diversicolor HIF-1α (HdHIF-1α) is 2833bp encoding a protein of 711aa and H. diversicolor HIF-1β (HdHIF-1β) is 1919bp encoding a protein of 590aa. Similar to other species' HIF-1, HdHIF-1 has one basic helix–loop–helix (bHLH) domain and two Per-Arnt-Sim (PAS) domains, and HdHIF-1α has a oxygen-dependent degradation domain (ODDD) with two proline hydroxylation motifs and a C-terminal transactivation domain (C-TAD) with an asparagine hydroxylation motif. Under normoxic conditions, HdHIF-1α and HdHIF-1β mRNAs were constitutively present in all examined tissues. Under hypoxia (2.0mg/L DO at 25°C) stress, HdHIF-1α expression was up-regulated in gills at 4h, 24h and 96h, and in hemocytes at 24h and 96h, while HdHIF-1β remained relatively constant. Under thermal stress (31°C), HdHIF-1α expression was significantly increased in gills at 4h, and hemocytes at 0h and 4h, while HdHIF-1β expression still remained relatively constant. These results suggested that HIF-1α may play an important role in adaption to poor environment in H. diversicolor. [ABSTRACT FROM AUTHOR]

The article discusses the use of loop-mediated isothermal amplification (LAMP) to detect abalone shrivelling syndrome-associated virus. It provides an overview about the marine gastropod mollusc Haliotis diversicolor Reeve and LAMP. It also suggests that the LAMP method was successfully modified in the study for AbSV detection, demonstrating high specificity and sensitivity at a level similar to other reported PCR methods for viral detection in molluscs.

The intracellular ice formation (IIF) behavior of Haliotis diversicolor (small abalone) eggs is investigated in this study, in relation to controlling the cooling rate and the concentration of dimethyl sulfoxide (DMSO). The IIF phenomena are monitored under a self-developed thermoelectric cooling (TEC) cryomicroscope system which can achieve accurate temperature control without the use of liquid nitrogen. The accuracy of the isothermal and ramp control is within ±0.5°C. The IIF results indicate that the IIF of small abalone eggs is well suppressed at cooling rates of 1.5, 3, 7 and 12°C/min with 2.0, 2.5, 3.0 and 4.0M DMSO in sea water. As 2.0M DMSO in sea water is the minimum concentration that has sufficient IIF suppression, it is selected as the suspension solution for the cryopreservation of small abalone eggs in order to consider the solution’s toxicity effect. Moreover, IIF characteristics of the cumulative probability of IIF temperature distribution are shown to be well fitted by the Weibull probabilistic distribution. According to our IIF results and the Weibull distribution parameters, we conclude that cooling at 1.5°C/min from 20 to −50°C with 2.0M DMSO in sea water is more feasible than other combinations of cooling rates and DMSO concentrations in our experiments. Applying this protocol and observing the subsequent osmotic activity, 48.8% of small abalone eggs are osmotically active after thawing. In addition, the higher the cooling rate, the less chance of osmotically active eggs. A separate fertility test experiment, with a cryopreservation protocol of 1.5°C/min cooling rate and 2.0M DMSO in sea water, achieves a hatching rate of 23.7%. This study is the first to characterize the IIF behavior of small abalone eggs in regard to the cooling rate and the DMSO concentration. The Weibull probabilistic model fitting in this study is an approach that can be applied by other researchers for effective cryopreservation variability estimation and analysis. [ABSTRACT FROM AUTHOR]

Abstract: Ubiquitin-conjugating enzymes (UBE2s or E2s) are characterized by the presence of a highly conserved ubiquitin-conjugating (UBC) domain, which predominantly determines the type of ubiquitin chains and directly controls the cellular fate of the substrate. In this study, an E2 homolog was identified and functionally characterized in abalone, which we named ab-UBE2D. The full-length cDNA consists of 1005 bp with an ORF encoding a protein of 147 amino acids. The deduced amino acid sequence shows ab-UBE2D shares conserved UBC domain with other E2 proteins and belongs to class I E2 enzyme family, which are further confirmed by phylogenetic tree analysis. Real-time PCR and western blot analyses showed that ab-UBE2D was ubiquitously expressed in abalone and the expression level of ab-UBE2d was significantly induced by LPS and Poly (I:C). Immunofluorescence microscopy staining demonstrated that native ab-UBE2D was mainly distributed in the cytoplast. Ubiquitination assay showed that ab-UBE2D had ubiquitin conjugating activity to form the enzyme-(Ub)n conjugates. Taken together, these results strongly suggest that ab-UBE2D is an E2 homolog and it may be involved in the immune response of abalone, Haliotis diversicolor supertexta. [Copyright &y& Elsevier]

Abstract: The Pacific abalone Haliotis diversicolor supertexta, of the family Haliotidae, is one of the most important shellfish species in eastern Asia. In Korea, it is intensively caught in Jeju Island, where it is highly appreciated as seafood. However, its natural population has declined drastically in the past several decades. To investigate its genetic characteristics for conservation and management purposes, the genetic variation in four populations from Jeju Island, Korea was analyzed using 20 microsatellite loci. All populations showed very high genetic diversity and a deficit of heterozygotes. The minor but significant genetic differentiations (both F ST and R ST) observed were mainly due to genetic differences between populations from Pyoson and other localities, suggesting that abalone may not be panmictic. The Pyoson abalone population showed a significant difference in allelic richness that could be related to harvesting intensity and stock enhancement activity. This result was also corroborated by an analysis of molecular variance (AMOVA). Overall, this report presents novel observations of the wild genetic population structure in H. diversicolor supertexta from Korea using microsatellite markers and provides critical information for supporting conservation strategies for this species, namely that the Pyoson abalone population should have a higher conservation priority due to its unique genetic characteristics. [Copyright &y& Elsevier]

ABSTRACT Analyses of lipid profile and fatty acid compositions of muscle and viscera of two commercially cultured abalone, namely Haliotis discus hannai Ino and Haliotis diversicolor Reeve, have been carried out. The total lipid levels of the viscera of both abalone were much higher than those of muscle. Phospholipids were the predominant lipid components in muscle, whereas triacylglycerol in viscera. The major fatty acids identified in muscle and viscera were C16:0, C18:0, C20:4 n-6, C20:5 n-3 and C22:5 n-3. Polyunsaturated fatty acids (PUFAs) accounted for over 40% of total fatty acids, and C20:4 n-6 and C20:5 n-3 were the most abundant PUFAs. Moreover, different fatty acid compositions were found between the muscle and viscera. Compared with muscle, viscera contained higher percentage of 4,8,12-trimethyltridecanoic acid and C18-PUFA, but lower percentage of C22-PUFA and dimethyl acetals. This study would increase awareness of the nutritional value of these two abalone and also contribute some important information to abalone aquaculture. PRACTICAL APPLICATIONS Haliotis discus hannai Ino and Haliotis diversicolor Reeve are the main commercially farmed species of abalone and have become the most important species of molluscan mariculture in China and occupied the important position in terms of commercial value among farmed molluscan products. This study analyzed the lipid profile and fatty acid compositions of muscle and viscera of the above two abalone species. The results would provide some useful nutrition information for the consumption and aquaculture of abalone. Meanwhile, the viscera of abalone are rich in total lipids and polyunsaturated fatty acid, indicating that the viscera would have great potential of exploitation and comprehensive utilization in lipids. [ABSTRACT FROM AUTHOR]

Abstract: Sodium alginate extracted from brown algae was reported to enhance the immune response and resistance of fish and shrimp. In this study, survival rates of the abalone, Haliotis diversicolor supertexta, against Vibrio parahaemolyticus, and its non-specific immune parameters such as the total haemocyte count (THC), phenoloxidase (PO) activity, respiratory bursts, superoxide dismutase (SOD) activity, phagocytic activity, and clearance efficiency to V. parahaemolyticus by H. diversicolor supertexta were determined when abalone (4.5 ± 0.4 g) were fed diets containing sodium alginate at 0, 1.0, 2.0, and 3.0 g kg−1. Abalone fed a diet containing sodium alginate at 2.0 and 3.0 g kg−1 for 14 days and at 1.0 g kg−1 for 21 days had significantly higher survival rates than those fed the control diet after challenge with V. parahaemolyticus. The relative survival percentages of abalone fed the 1.0, 2.0, and 3.0 g kg−1 sodium alginate-containing diets for 14 and 21 days were 16.1%, 40.0%, and 48.0%, and 63.6%, 27.3% and 22.6%, respectively. The PO activity, respiratory bursts, SOD activity, and phagocytic activity and clearance efficiency of V. parahaemolyticus of abalone fed the sodium alginate-containing diets at 1.0, 2.0, and 3.0 g kg−1 were significantly higher than those of abalone fed the control diet for 14 days. After 21 days, the PO activity, respiratory bursts, SOD activity, and phagocytic activity and clearance efficiency of V. parahaemolyticus by abalone fed the sodium alginate-containing diet at 1.0 g kg−1 were significantly higher than those of abalone fed the other diets. It was concluded that sodium alginate can be used as an immunostimulant for abalone through dietary administration to enhance immune responses of abalone and resistance against V. parahaemolyticus, which were related to the dose and timing of administration. [Copyright &y& Elsevier]

Abstract: GABAA receptor-associated protein (GABARAP), a multifunctional protein participating in autophagy process, is evolutionarily conserved and involves in innate immunity in eukaryotic cells, but currently there is no research on the relationship between GABARAP and innate immunity in mollusc. In the present study, the GABARAP full-length cDNA and its genomic DNA were firstly cloned from small abalone (Haliotis diversicolor), which was named as saGABARAP. Its full-length cDNA is 963 bp with a 354 bp open reading frame encoding a protein of 117 aa, a 276 bp 5′-UTR, and a 333 bp 3′-UTR including a poly(A) tail, two typical polyadenylation signals (AATAA) and two RNA instability motifs (ATTTA). The deduced protein has an estimated molecular weight of 13.9 kDa and a predicted PI of 8.73. Its genomic DNA comprises 4352 bp, containing three exons and two introns. Quantitative real-time PCR analysis revealed that saGABARAP was constitutively expressed in all examined tissues, with the highest expression level in hepatopancreas, and was upregulated in hepatopancreas and hemocytes after bacterial challenge. In addition, saGABARAP was ubiquitously expressed at all examined embryonic and larval development stages. These results suggested that saGABARAP could respond to bacteria challenge and may play a vital role in the adult innate immune system against pathogens and the development process of abalone embryo and larvae. [Copyright &y& Elsevier]

Abstract: A 5781-base pair (bp) fragment of genomic DNA from the Taiwanese abalone herpesvirus was obtained and showed 99% (5767/5779) homology in the nucleotide sequence and 99% (1923/1926) in the amino acid sequence with the DNA polymerase gene of the abalone herpesvirus strain Victoria/AUS/2007. Homology of the amino acid sequence with the DNA polymerase of ostreid herpesvirus 1 was 30% (563/1856). In this study, a PCR-based procedure for detecting herpesvirus infection of abalone, Haliotis diversicolor supertexta, in Taiwan was developed. The method employed primer sets targeting the viral DNA polymerase gene, and was able to amplify DNA fragments of the expected size from infected samples. Primer sets of 40f and 146r were designed for amplification of an expected PCR product of 606bp. Combining the new PCR protocol with histopathology, this assay can serve as a reliable diagnostic for herpesvirus infections in abalone. [Copyright &y& Elsevier]

Abstract: Interleukin receptor‐associated kinase (IRAK)-1 binding protein 1 (IRAK1BP1) is a critical factor in preventing dangerous overproduction of proinflammatory cytokines by the innate immune system and in influencing the specificity of TLR responses. In this study, a first molluscan IRAK1BP1 gene, saIRAK1BP1, was cloned from the small abalone (Haliotis diversicolor). Its full-length cDNA sequence is 1047bp, with a 747bp open reading frame encoding a protein of 249 aa. The molecular mass of the deduced protein is approximately 28.1kDa with an estimated pI of 8.87, and shows highest identity (52%) to acorn worm Saccoglossus kowalevskii. Amino acid sequence analysis revealed that saIRAK1BP1 shares a conserved SIMPL domain. Quantitative real-time PCR was employed to investigate the tissue distribution of saIRAK1BP1 mRNA, and its expression in abalone under bacteria challenge and larvae at different developmental stages. The saIRAK1BP1 mRNA could be detected in all examined tissues, with the highest expression level in hemocytes, and was up-regulated in gills, kidneys and hemocytes after bacteria injection. Additionally, saIRAK1BP1 was constitutively expressed at all examined developmental stages. These results indicate that saIRAK1BP1 play an important role in the adult abalone immune system and might be essential in embryo and larval development in abalone. [Copyright &y& Elsevier]

Abstract: Haliotis diversicolor (small abalone) is an important seafood found along the southern coast of China. Since 1999, the yields of cultured abalone in China have been severely affected by an epidemic of continuous outbreaks of a fatal disease. A novel double-stranded DNA virus, abalone shriveling syndrome-associated virus (AbSV), was proven to be one of the main causative agent. Although the pathogenicity and genome of AbSV has been ascertained, the epidemiology of AbSV remains to be investigated. In this study, four pairs of AbSV-specific primers were designed on the basis of the AbSV genome, and were tested for their specificities and sensitivities in quantitative real-time PCRs (qPCRs) after optimization of the annealing temperature. The 3F3/3B3 primer pair was finally chosen with a good specificity and high efficiency of amplification, with a detection limit of about 10 copies of recombinant plasmid containing AbSV genes in a 20-μL reaction mixture. In the detection of AbSV in abalone samples along the southern coast of China, most of the diseased samples had more than 80 virus copies in 1ng host genome DNA. AbSV was also demonstrated in mature hybrid (LY) and juvenile (JH) abalones from assays of healthy animals collected in recent years. [Copyright &y& Elsevier]

Abstract: Haliotis diversicolor (small abalone) is an economic seafood found off the Southern coast of China. Since 1999, the cultured abalone yields in China have been affected severely by continual outbreaks of a fatal epidemic disease caused by abalone shriveling syndrome associated virus (AbSV), a double-stranded DNA virus. Although the pathogenicity and genome of AbSV have been ascertained, the epidemiology of AbSV infection remains to be investigated. In the present study, four pairs of AbSV-specific primers were designed on the basis of open reading frame (ORF)24 and ORF25 sequences in the AbSV genome. Two nested PCR detection methods were established by optimization of the annealing temperatures of primers. The results showed that the specificity of primers for AbSV detection could not be interfered with by the host genome and other aquaculture species or viruses. The detection limits of the two methods were about 10 copies of recombinant plasmid containing AbSV genes in 20μL reaction mixture. The results of detection of the AbSV epidemic showed that AbSV was still present in juvenile abalones in some farms along the Southern coast of China (Fujian and Guangdong). [Copyright &y& Elsevier]

The small abalone, Haliotis diversicolor supertexta, of the family Haliotidae, is one of the most important species of marine shellfish in eastern Asia. Over the past few decades, this species has drastically declined in Korea. Thus, hatchery-bred seeds have been released into natural coastal areas to compensate for the reduced fishery resources. However, information on the genetic background of the small abalone is scarce. In this study, 20 polymorphic microsatellite DNA markers were identified using next-generation sequencing techniques and used to compare allelic variation between wild and released abalone populations in Korea. Using high-throughput genomic sequencing, a total of 1516 (2.26%; average length of 385 bp) reads containing simple sequence repeats were obtained from 86,011 raw reads. Among the 99 loci screened, 28 amplified successfully, and 20 were polymorphic. When comparing allelic variation between wild and released abalone populations, a total of 243 different alleles were observed, with 18.7 alleles per locus. High genetic diversity (mean heterozygosity = 0.81; mean allelic number = 15.5) was observed in both populations. A statistical analysis of the fixation index (FST) and analysis of molecular variance (AMOVA) indicated limited genetic differences between the two populations (FST = 0.002, p > 0.05). Although no significant reductions in the genetic diversity were found in the released population compared with the wild population (p > 0.05), the genetic diversity parameters revealed that the seeds released for stock abundance had a different genetic composition. These differences are likely a result of hatchery selection and inbreeding. Additionally, all the primer pair sets were effectively amplified in another congeneric species, H. diversicolor diversicolor, indicating that these primers are useful for both abalone species. These microsatellite loci may be valuable for future aquaculture and population genetic studies aimed at developing conservation and management plans for these two abalone species. [ABSTRACT FROM AUTHOR]

Abstract: Insulin-like growth factor binding protein 7 (IGFBP7), the only member of the IGFBP superfamily that binds strongly to insulin, may have different functions from other IGFBPs. Unlike other IGFBPs, there is no knowledge available on aquatic invertebrate IGFBP7. In this study, a molluscan IGFBP7 gene, saIGFBP7, was cloned for the first time from the small abalone Haliotis diversicolor. Its full-length cDNA sequence is 1812bp, with a 720bp open reading frame encoding a protein of 239 aa. The molecular mass of the deduced protein is approximately 25.37kDa with an estimated pI of 5.00, and it shares highest 41% identity to IGFBP7 of Amblyomma americanum. Analysis of conserved domains revealed the presence of an IGFBP N-terminal domain (IB), a kazal-type serine proteinase inhibitor domain (KI), and an immunoglobulin-like C2 domain (IgC2) in saIGFBP7. Furthermore, the 12 cysteine residues and the signature amino acid motif ‘xCGCCxxC’ which are characterized by the amino terminus region of the IGFBP superfamily are all presented in saIGFBP7. Quantitative real-time PCR and western blot were employed to investigate the tissue distribution of saIGFBP7, and its expression under bacterial challenge. The saIGFBP7 mRNA and protein could be detected in all examined tissues, with the highest expression level in hemocytes, higher expression level in gills, and was up-regulated in hemocytes and gills after bacterial injection. In addition, saIGFBP7 mRNA transcripts were observed in a subset of the branchial epithelium and the nucleus of hemocytes using the in situ hybridization method. Interestingly, saIGFBP7 was detected mainly in the goblet-like cell of the branchial epithelium by immunohistochemistry. These results suggested that saIGFBP7 was likely to be involved in a function associated with pathogenic infection and may play an important role in the adult abalone immune system. [Copyright &y& Elsevier]

Abstract: The gut of abalone contains a diverse population of bacteria which contribute to food digestion and nutrient absorption. To investigate the development of gut microflora in the small abalone Haliotis diversicolor and the effect of various diets on the bacterial communities, we analyzed the composition of gut microbial flora and the bacterial digestive enzymes in connection with four important development stages, i.e., larva, early-juvenile, late-juvenile and grow-out. Among these cultivated bacteria, the Firmicutes group was isolated from both the larval and early-juvenile stages; the CFB (Cytophaga/Flexibacteria/Bacterioides) group was detected in early juveniles, late juveniles and grow-out; the γ-proteobacteria existed throughout the four development stages. The isolated Bacillus sp. and Pseudoalteromonas sp. dominated in the larvae and were rarely detected in other stages. Subsequently, the genus Vibrio predominated in the latter three phases after the first feeding. Cluster analysis of the DGGE (Denaturing Gradient Gel Electrophoresis) patterns was basically congruent with the result of culture-dependent method in which the gut bacterial community indicated the successional change in the development stages. And the artificial diets contribute to the change of gut bacterial composition to some extent. The enzyme assay showed that the gut bacteria isolated from the early juveniles produced the greatest diverse digestive enzymes compared with those isolated from other development stages. Most cultivated bacteria in each development stage showed protease, lipase or alginate-degrading activities which could help the farmed abalone H. diversicolor to digest diets. All results showed that a unique but interrelated bacterial flora was developed in the abalone gut which was coincident with the change of diets. [Copyright &y& Elsevier]