Cryopreservation of sperm from farmed Pacific abalone,Haliotis discus hannai

This study aimed to improve a sperm cryopreservation protocol for farmed Pacific abalone, Haliotis discus hannai. Dimethyl sulfoxide (Me₂SO), glycerol, ethylene glycol (EG), propylene glycol (PG), and methanol were chosen as cryoprotectants (CPAs). Four different equilibration time (5, 10, 30, and 60 min), and two types of equilibration temperature (4 °C and 20 °C) were selected at the present experiment. Most equilibration temperatures with each CPA showed significant differences among different equilibration time. Post-thaw sperm motility of five CPAs showed no significant difference at two equilibration temperature. Based on these results, 8% Me₂SO, 8% EG, 6% PG, 2% glycerol, and 2% methanol were chosen to determine optimal conditions for sperm cryopreservation of H. discus hannai. The highest post-thaw sperm motility (8% Me₂SO: 50.6%, 8% EG: 45.6%, 2% glycerol: 44.5%, 6% PG: 28.7%, 2% methanol: 25.4%) was achieved after exposing sperm to liquid nitrogen (LN₂) vapor for 10 min at 5 cm above the LN₂ surface and then submerging them in LN₂ for at least 2 h followed by thawing at 60 °C with seawater and recovering them at 20 °C with seawater. In this study, 8% Me₂SO and 2% glycerol were chosen to check post-thaw sperm quality to estimate percentages of plasma membrane integrity (PMI), mitochondrial potential analysis (MP), and acrosome integrity (AI) using fluorescent techniques. No significant difference in PMI, MP, and AI was found between sperm cryopreserved with 8% Me₂SO and those cryopreserved with 2% glycerol. The current study has demonstrated that 8% Me₂SO was optimal for sperm cryopreservation for H. discus hannai with 5 min of equilibration time, 5 cm of rack height and 60 °C of thawing temperature. The present research provides more effective cryopreservation methods for H. discus hannai sperm than previous studies.     

cDNA cloning of an alginate lyase from abalone, Haliotis discus hannai

An alginate lyase, termed HdAly in the present paper, was isolated from the hepatopancreas of abalone, Haliotis discus hannai, by ammonium sulfate fractionation, followed by TOYOPEARL CM-650M column chromatography. Enzymatic properties of HdAly were similar to those of previously reported Haliotis and Turbo poly(M) lyases, e.g., it preferentially degraded a poly(beta-D-mannuronate)-rich substrate with an optimal pH and temperature at pH 8.0 and 45 degrees C, respectively. In order to determine the primary structure of abalone lyase that is still poorly understood, cDNAs for HdAly were cloned by PCR from the abalone hepatopancreas cDNA library and sequenced. From the nucleotide sequences of the cDNAs, the sequence of 909 bp in total was determined, and the amino acid sequence of 273 residues was deduced from the translational region of 822 bp locating at nucleotide positions 27-848. The N-terminal region of 16 residues, except for the initiation Met in the deduced sequence, was regarded as the signal peptide since it was absent in the HdAly protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretary proteins. This suggests that HdAly is initially produced as a precursor possessing the signal peptide in hepatopancreatic cells and then secreted into digestive tract as the mature form. Thus, the mature HdAly was regarded to consist of 256 residues with the calculated molecular mass of 28895.5 Da. The amino acid sequence of HdAly showed 85 and 28% identity to those of Turbo cornutus alginate lyase SP2 and the C-terminal region of Chlorella virus lyase-like protein CL2, respectively, while it showed no significant identity to those of any bacterial alginate lyases. In order to provide the basis for the structure-function studies and various applications of the abalone lyase, a bacterial expression system was constructed by means of the HdAly-cDNA and pET-3a expression plasmid. Although the active recombinant HdAly was hardly produced at a cultivation temperature 37 degrees C in Escherichia coli BL21 (DE3), a small amount of soluble and active enzyme could be produced when the temperature was lowered to 19 degrees C.     

Metabolic responses to dietary cholecalciferol and phosphorus in abalone Haliotis discus hannai ino

Metabolic responses of cholecalciferol (VD(3)) and minerals (Ca, P and Mg) in abalone Haliotis discus hannai Ino to dietary VD(3) and phosphorus (P) were investigated. Based on a 2 x 2 factorial design, four casein-gelatin-based diets were formulated. The basal diet was supplemented with either 0 or 2000 IU VD(3)/kg diet and 0 or 10 g P/kg diet. The abalone was reared in P-free artificial seawater for 55 days. Results showed that dietary VD(3) was hydroxylated to 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25(OH)(2)D(3)] in abalone, and subsequently raised the serum levels of these two VD(3) metabolites. Dietary P deficiency elevated serum 1 alpha,25(OH)(2)D(3) level only when the dietary VD(3) supplementation was sufficient. The supplementations of either dietary VD(3) or P significantly increased the levels of P in serum, mantle and hepatopancreas, and only the addition of VD(3) significantly raised the concentrations of Ca in serum and mantle (P<0.05). Interaction between dietary VD(3) and P was only found significant on the concentrations of P and Mg in mantle (P<0.05). The concentrations of Ca, P and Mg in muscle were not significantly influenced by these dietary treatments. Hence, the metabolic responses in serum, muscle, mantle and hepatopancreas of abalone to dietary VD(3) and P were in different manners.     

Physiological performance of juvenile Haliotis rufescens and Haliotis discus hannai abalone exposed to the withering syndrome agent

The presence of Wolbachia and Cardinium bacteria has been documented in many arthropod species, including the predatory mite Metaseiulus (=Typhlodromus or Galendomus) occidentalis (Nesbitt) (Acari: Phytoseiidae). We show that Tetranychus urticae, the prey of Metaseiulus occidentalis, contains Wolbachia and no detectable Cardinium using quantitative PCR (qPCR). Starvation for 72 h at 22°C eliminated most, if not all, Wolbachia in M. occidentalis adult females from 7 laboratory colonies. Refeeding of M. occidentalis with T. urticae after starvation for 72 h restored the amounts of Wolbachia in M. occidentalis to those of prestarvation levels, suggesting that Wolbachia detected in M. occidentalis starved for shorter periods of time in current, and some previous, studies likely came from T. urticae. Furthermore, eggs from all M. occidentalis colonies examined were free of Wolbachia if they were surface-decontaminated with 0.3% sodium hypochlorite before DNA extraction. Cardinium was present in 6 of 14 laboratory colonies of M. occidentalis. Starvation for 3, 24, 48, and 72 h had no effect on the amounts of Cardinium in adult females from the Cardinium-positive colonies. Eggs from these colonies were positive for Cardinium but contained less than 1% of the titers found in adult females. The data suggest that Cardinium, but not Wolbachia, is an endosymbiont in certain populations of M. occidentalis. In light of our current findings, we recommend specific practices for the identification of potential symbionts in predatory arthropod species using the PCR.     

Purification and cDNA cloning of a cellulase from abalone Haliotis discus hannai.

A cellulase [endo-beta-1,4-D-glucanase (EC 3.2.1.4)] was isolated from the hepatopancreas of abalone Haliotis discus hannai by successive chromatographies on TOYOPEARL CM-650M, hydroxyapatite and Sephacryl S-200 HR. The molecular mass of the cellulase was estimated to be 66 000 Da by SDS/PAGE, thus the enzyme was named HdEG66. The hydrolytic activity of HdEG66 toward carboxymethylcellulose showed optimal temperature and pH at 38 degrees C and 6.3, respectively. cDNAs encoding HdEG66 were amplified by the polymerase chain reaction from an abalone hepatopancreas cDNA library with primers synthesized on the basis of partial amino-acid sequences of HdEG66. By overlapping the nucleotide sequences of the cDNAs, a sequence of 1898 bp in total was determined. The coding region of 1785 bp located at nucleotide position 56-1840 gave an amino-acid sequence of 594 residues including the initiation methionine. The N-terminal region of 14 residues in the deduced sequence was regarded as the signal peptide as it was absent in HdEG66 protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretory proteins. Thus, matured HdEG66 was thought to consist of 579 residues. The C-terminal region of 453 residues in HdEG66, i.e. approximately the C-terminal three quarters of the protein, showed 42-44% identity to the catalytic domains of glycoside hydrolase family 9 (GHF9)-cellulases from arthropods and Thermomonospora fusca. While the N-terminal first quarter of HdEG66 showed 27% identity to the carbohydrate-binding module (CBM) of a Cellulomonas fimi cellulase, CenA. Thus, the HdEG66 was regarded as the GHF9-cellulase possessing a family II CBM in the N-terminal region. By genomic PCR using specific primers to the 3'-terminal coding sequences of HdEG66-cDNA, a DNA of 2186 bp including three introns was amplified. This strongly suggests that the origin of HdEG66 is not from symbiotic bacteria but abalone itself.     

皱纹盘鲍的个体能量收支

对皱纹盘鲍的呼吸、摄食、生长及能量收支等实验研究表明, 鲍的耗氧率与壳长、体重、温度及昼夜变化有关, 耗氧率与壳长、体重均呈幂函数关系, 一天中16~4时(夜间)耗氧率高于4~16时(白天)且在18~20时达峰值.同温度下鲍日摄食率与体重呈幂指数关系, 日(相对)摄食率随温度升高而增加, 而日相对摄食率、日相对生长率均随壳长、体重增加呈下降趋势.鲍在14~20℃内对海带的总转化效率为53%.鲍软体部、海带及鲍粪便干品的比能值分别为19.2、8.57和7.23kJ·g^-1.14~20℃皱纹盘鲍摄入能量的34.6~48.6%为粪能, 22.0~38.2%的能量用于自身代谢, 5.6~28.2%用于贝体软体部的生长.     

Effects of dietary pyridoxine on immune responses in abalone, Haliotis discus hannai Ino

A feeding experiment was conducted to investigate the effects of dietary pyridoxine (PN) on the immune responses of abalone, Haliotis discus hannai Ino. Purified diets supplemented with 0, 40, 800 mg PN kg(-1) or 80 mg kg(-1) of 4-deoxypyridoxine (PN antagonist) were fed to adult abalone (initial weight 45.77 +/- 0.25 g; initial shell length 68.02 +/- 0.78 mm) for 90 days. The air-dried brown kelp, Laminaria japonica, was used as a control diet. Each diet was fed to three replicate groups of abalone in a recirculation system using a completely randomised design. The results showed that weight gain ratio (WGR) of the abalone generally increased with the level of dietary PN supplementation though no significant differences were found among the treatments (P > 0.05). Phagocytic and phenoloxidase activities were significantly higher in abalone fed diets supplemented with 800 mg PN kg(-1) than those fed the PN-free diet or the one with 4-deoxypyridoxine (P < 0.05). Agglutination titre and respiratory burst activity were significantly higher in abalone fed diets supplemented with 40 mg PN kg(-1) than those fed the PN-free diet or the one with 4-deoxypyridoxine (P < 0.05). There were no significant differences in immunological characteristics between the abalone fed the diet containing 40 mg PN kg(-1) and those fed the diet containing 800 mg PN kg(-1) (P > 0.05). L. japonica resulted in significantly lower agglutination titre, respiratory burst and phagocytic activities than the artificial diets supplemented with 40 or 800 mg PN kg(-1) (P < 0.05). Total haemocyte count (THC), serum protein concentration, and the activities of lysozyme and acid phosphatase were not significantly affected by the dietary treatments (P > 0.05). These results demonstrate that dietary deficiency of pyridoxine suppresses the immune functions in H. discus hannai, and further investigations are needed to optimise the dietary level of this vitamin for maintaining the best immune responses in abalone.     

Isolation and characterization of microsatellite DNA markers in the Pacific abalone,Haliotis discus hannai

We developed 17 new microsatellite markers in Haliotis discus hannai. All loci were found to be polymorphic with an average of 13.1 alleles per locus (range 3–28). The mean observed and expected heterozygosities were 0.77 (range 0.17–1.00) and 0.79 (range 0.42–0.96), respectively. Six loci deviated significantly from Hardy–Weinberg proportions, and thus should be used with caution. The high variabilities revealed in this study suggest that these microsatellite loci should provide useful markers for studies of trait mapping, kinship and population genetics.     

皱纹盘鲍的个体能量收支

对皱纹盘鲍的呼吸、摄食、生长及能量收支等实验研究表明, 鲍的耗氧率与壳长、体重、温度及昼夜变化有关, 耗氧率与壳长、体重均呈幂函数关系, 一天中16~4时(夜间)耗氧率高于4~16时(白天)且在18~20时达峰值.同温度下鲍日摄食率与体重呈幂指数关系, 日(相对)摄食率随温度升高而增加, 而日相对摄食率、日相对生长率均随壳长、体重增加呈下降趋势.鲍在14~20℃内对海带的总转化效率为53%.鲍软体部、海带及鲍粪便干品的比能值分别为19.2、8.57和7.23kJ·g^-1.14~20℃皱纹盘鲍摄入能量的34.6~48.6%为粪能, 22.0~38.2%的能量用于自身代谢, 5.6~28.2%用于贝体软体部的生长.     

Ovarian follicle cells are the site of vitellogenin synthesis in the Pacific abalone Haliotis discus hannai

Only a few biochemical and molecular studies on yolk proteins (vitellins) have been carried out in mollusks, mainly in bivalves, while information on prosobranch vitellogenesis is still limited. In this study, we cloned a full-length cDNA encoding vitellogenin (Vg) in the Pacific abalone Haliotis discus hannai. The complete Vg cDNA consists of 7753 nucleotides with a long open reading frame encoding 2391 amino acid residues. The deduced primary structure contains the N-terminal amino acid sequences of the 95 kDa and 150 kDa subunits of vitellin of the abalone and shows similarities to Vgs of other mollusk, fish, nematode and coral species. In common with bivalve Vgs, the abalone Vg gene was expressed only in the ovary. In situ hybridization analysis further localized Vg mRNA to the follicle cells in the ovary. We conclude that the follicle cells are the site of Vg synthesis in H. discus hannai.     

Development of 15 polymorphic genic microsatellite DNA markers of Pacific abalone Haliotis discus hannai

Fifteen polymorphic genic microsatellite DNA markers were developed based on the expressed sequence tags of Pacific abalone Haliotis discus hannai we ourselves generated, which were used to type 32 individuals representing a cultured population. The number of alleles each locus ranged from two to 12. The observed and expected heterozygosities ranged from 0.094 to 0.969 and from 0.091 to 0.878, respectively. Among 15 loci, four were found to deviate significantly from Hardy–Weinberg equilibrium (P_HW < 0.001). No linkage disequilibrium was found between these loci. It is certain that these markers will facilitate the management and exploitation of the genetic resource of Pacific abalone.     

Isolation and cloning of an endo-beta-1,4-mannanase from Pacific abalone Haliotis discus hannai

An endo-beta-1,4-mannanase was isolated from digestive fluid of Pacific abalone, Haliotis discus hannai, by successive chromatographies on TOYPEARL CM-650M, hydroxyapatite, and TOYOPEARL HW50F. The abalone mannanase, named HdMan in the present paper, showed a molecular mass of approximately 39,000 Da on SDS-PAGE, and exhibited high hydrolyic activity on both galactomannan from locust bean gum and glucomannan from konjac at an optimal pH and temperature of 7.5 and 45 degrees C, respectively. HdMan could degrade either beta-1,4-mannan or beta-1,4-mannooligosaccharides to mannotriose and mannobiose similarly to beta-1,4-mannanases from Pomacea, Littorina, and Mytilus. In addition, HdMan could disperse the fronds of a red alga Porphyra yezoensis into cell masses consisting of 10-20 cells that are available for cell engineering of this alga. cDNAs encoding HdMan were amplified by polymerase chain reaction from an abalone-hepatopancreas cDNA library. From the nucleotide sequences of the cDNAs, the sequence of 1232 bp in total was determined and the amino-acid sequence of 377 residues was deduced from the translational region of 1134 bp locating at nucleotide positions 15-1148. The N-terminal region of 17 residues except for the initiation Met, was regarded as the signal peptide of HdMan because it was absent in the HdMan protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretory proteins. Accordingly, mature HdMan was considered to consist of 359 residues with the calculated molecular mass of 39,627.2 Da. HdMan is classified into glycoside hydrolase family 5 (GHF5) on the basis of sequence homology to GHF5 enzymes.     

Effects of traditional Chinese medicine on immune responses in abalone, Haliotis discus hannai Ino

A traditional Chinese medicine (TCM) preparation was formulated from orange peel (Pericarpium Citri Reticulatae), hawthorn (Crataegus pinnatifida), astragalus (Astragalus membranaceus (Fisch.) Bunge), pilose asiabell root (Radix codonopsis), indigowoad root (Radix isatidis), taraxacum (Herba taraxaci) and malt (Fructus Hordei Germinatus) at a weight ratio of 1:1:1.5:1.5:1.5:1.5:2. A feeding experiment was conducted to determine the effects of TCM on innate immunity of abalone, Haliotis discus hannai Ino. Artificial diets containing 1%, 3%, 5% TCM preparation, 1% hawthorn or 1% astragalus, respectively, were fed to juvenile abalone (initial weight 10.38+/-2.51 g; initial shell length 44.15+/-4.15 mm) for 80 days. A TCM-free diet was used as a control. Each diet was fed to three replicate groups of abalone using a randomized design. The results indicated that phagocytic activity was significantly higher in abalone fed 3%, 5% TCM preparation, 1% astragalus or 1% hawthorn (P<0.05). Respiratory burst activity was significantly higher in abalone fed 1%, 3%, 5% TCM preparation, 1% astragalus or 1% hawthorn (P<0.05). Agglutination titre was significantly higher in abalone fed 5% TCM preparation (P<0.05). Weight gain ratio (WGR), daily increment in shell length (DISL), total haemocyte count (THC), plasma protein concentration, and the activity of acid phosphatase (ACP) were not significantly affected by the TCM preparation (P>0.05). These results indicate that TCM preparation can modulate the immunity of H. discus hannai, and it is very possible that TCM might be used as immunostimulants in abalone farming.     

Development of gene-targeted SNP markers for genomic mapping in Pacific abalone Haliotis discus hannai Ino

Useful and novel DNA markers are needed for aquaculture genetics and breeding. In this study, we report the discovery and development of gene-targeted single nucleotide polymorphisms (SNPs) for genomic mapping in the Pacific abalone Haliotis discus hannai Ino. Single EST or EST-contigs from 66 genes that had positive BLASTx matches (E-value ≤ 1e-8) were used for polymerase chain reaction (PCR) amplification. PCR products from the two parents of one mapping family were directly sequenced, and 83 SNP loci were found from 17 genes. Allele-specific PCR (AS-PCR) was developed and optimized for genotyping of 11 SNP loci in 120 progeny of the mapping family. Nine of the loci conformed to the expected Mendelian ratio of 1:1 based on the χ² test (P > 0.05) and could potentially be used for linkage map construction. Our data also indicate that the sequencing of two parents may be a practical strategy for the discovery of informative SNPs for linkage mapping in a particular mapping population.     

Proteomic analysis of muscle between hybrid abalone and parental lines Haliotis gigantea Reeve and Haliotis discus hannai Ino

To understand the potential molecular mechanism of heterosis, protein expression patterns were compared from hybrids of Haliotis gigantea (G) and Haliotis discus hannai (D) using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight analyses. Expression differences were observed in muscle samples from the four groups with 673±21.0 stained spots for H. discus hannai ♀ × H. discus hannai ♂ (DD), 692±25.6 for H. gigantea ♀ × H. gigantea ♂ (GG), 679±16.2 for H. discus hannai ♀ × H. gigantea ♂ (DG) (F₁ hybrid) and 700±19 for H. gigantea ♀ × H. discus hannai ♂ (GD) (F₁ hybrid). Different 2-DE image muscle protein spots had a mirrored relationship between purebreds and the F₁ hybrid, suggesting that all stained spots in F₁ hybrid muscle were on 2-DEs from parents. DD and DG clustered together first, and then clustered with GD, whereas the distance of DD and GG was maximal according to hierarchical cluster analysis. We identified 136 differentially expressed protein spots involved in major biological processes, including energy metabolism and stress response. Most energy metabolism proteins were additive, and stress-induced proteins displayed additivity or over-dominance. In these 136 identified protein spots, hybrid offspring with additivity or over-dominance accounted for 68.38%. Data show that a proteomic approach can provide functional prediction of abalone interspecific hybridization.     

Characterization of 12 single nucleotide polymorphisms (SNPs) in Pacific abalone, Haliotis discus hannai

We report here for the first time 12 polymorphic single nucleotide polymorphisms (SNPs) in a commercially important gastropod, Pacific abalone (Haliotis discus hannai) that were identified by searching expressed sequence tag database. These SNP loci (seven nuclear and five mitochondrial SNPs) were polymorphic among 37 wild abalone individuals, based on a four-primer allele-specific polymerase chain reaction analysis. All loci had two alleles and the minor allele frequency ranged from 0.027 to 0.473. For the seven nuclear SNPs, the expected and observed heterozygosities ranged from 0.053 to 0.499 and from 0.054 to 0.811, respectively.     

Expression characterization and activity analysis of a cathepsin B from Pacific abalone Haliotis discus hannai

Cathepsin B (EC 3.4.22.1) is a member of lysosomal cysteine protease and has a papain-like fold. In mammals, it is involved in protein degradation and other physiological processes including immune response. However, little is known about the function of cathepsin B in mollusks. In this study, we identified and analyzed a cathepsin B homolog (HdCatB) from Pacific abalone (Haliotis discus hannai), an economically important mollusk species cultured in East Asia. HdCatB is composed of 336 amino acid residues and its mature form is predicted to start at residue 86. HdCatB possesses typical domain architecture of cathepsin B and contains a propeptide region and a cysteine protease domain, the latter containing the four active site residues (Q108, C114, H282, and N302) that are conserved in many different organisms. HdCatB shares 40–60% overall sequence identities with the cathepsin Bofa number of vertebrates and invertebrates and is phylogenetically very close to mollusk cathepsin B. Quantitative real time RT-PCR analysis revealed that HdCatB expression occurred in multiple tissues and was upregulated by bacterial infection. Recombinant HdCatB purified from Escherichia coli exhibited apparent protease activity, which was optimal at 45 °C and pH 6.0. These results indicate that HdCatB is a bioactive protease that is likely to be implicated in the immune response of abalone during bacterial infection.