Aminopeptidase Play a Critical Role in the Accumulation of Free Amino Acids in Abalone(Haliotis discus hannai)During Cold Storage

Abalones reveal unique taste after processing,mainly because of their abundant free amino acids(FAAs)and nucleotides.FAAs are nutrition components that can contribute to the unique taste.However,which factor(s)is responsible for the accumulation of FAAs still need further studies.To analyze the production of FAAs,we studied the variation of FAAs during 7 days of storage at 4℃.The content of taste-active amino acids,including Asp,Glu,Ser,and Gly increased by 1.7-fold,2.0-fold,3.0-fold,and 8.4-fold,respectively.The relative activity of cathepsin L and aminopeptidase(AP)increased significantly during the cold storage period.To identify AP in abalone and its function in mediating the production of FAAs,an aminopeptidase with wide substrate specificity was then extracted and purified from abalone muscle to homogeneity.Purified AP with a molecular mass of 100 kDa exhibited its maximum activity at 30℃,pH 7.5,and was further confirmed by LC-MS.Bestatin specifically inhibited the activity of AP,and metalloproteinase inhibitors EDTA,EGTA and 1,10-phenanthroline also suppressed its activity to different degrees.Based on its highest activity to substrate Leu-MCA and its peptide sequences,the purified enzyme was identified as leucine aminopeptidase(LAP).Our present study indicated the essential role of AP for FAAs accumulation during cold storage of abalone.

Structural characterization and immunostimulatory activity of a water-soluble polysaccharide from abalone(Haliotis discus hannai Ino)muscle

A water-soluble polysaccharide from abalone muscle(AMPP)was isolated.The contents of carbohydrate,protein,uronic acid,and sulfate in AMPP were 83.5%,0.5%,2.7%,and 2.6%,respectively.High-performance liquid chromatography analysis indicated that AMPP was homogeneous and had an average molecular weight of approximately 3.2 kDa.The main monosaccharides of AMPP were glucose(Glc)and mannose with a molar ratio of 99.7:0.3.The structural characteristics of AMPP were elucidated through methylation analysis,Fourier transform infrared spectroscopy,and nuclear magnetic resonance spectroscopy.The linkages of AMPP consisted of terminal,1,4-linked,1,6-linked,and 1,4,6-linked Glcp with a molar ratio of 3.1:7.2:1.0:2.5.In one repeat unit of the proposed AMPP structure,the backbone chain was composed of eight 1→4 glycosidic bonds and one 1→6 glycosidic bond,with three branch chains linked by 1→6 glycosidic bond.In addition,AMPP was found to possess potent immunostimulatory activity via rising phagocytosis of RAW264.7 cells and promoting secretion of TNF-α.

Biological Characteristics and Pathogenicities of Shewanella algae and Shewanella abalone from Babylonia

[Objective] The biological characteristics and pathogenicities of Shewanella algae and Shewanella abalone from Babylonia were studied in this paper. [Method]The hemolytic bacteria were isolated from the hepatopancreas of Babylonia suffered from proboscis edema with blood agar plate. The dominant bacterial community in the ill Babylonia was identified by 16 S r DNA sequence analysis, and the bacterial cultural and biochemical characteristics and pathogenicities were studied. [Result]The Shewanella bacteria, including Shewanella algae and Shewanella abalone, are the dominant bacterial community in Babylonia suffered from proboscis edema.The colony characteristics of Shewanella algae in nutrient agar medium, TCBS agar medium and CHROMagar vibrio colored medium were similar to those of Shewanella abalone. Shewanella algae possessed β-hemolysis and Shewanella abalone possessed α-hemolysis in the blood agar plate. The biochemical reaction of Shewanella algae and Shewanella abalone was all of non-fermentation type. The results of artificial infection test showed that half lethal dose（LD50） of the test strains of Shewanella algae was 10-5.50/0.1 ml. The test strains of Shewanella algae have strong toxicity, and could cause mice and chickens to die of sepsis with mortality of100%. The mortality of Babylonia infected with Shewanella algae was 10%; while the survived Babylonia lost the ability of moving and intaking for a long time, but they were not suffered from proboscis edema. There was no death in mice or chicks infected with Shewanella abalone, but their livers and spleens were slightly hyperemic and swelling. There was also no death in Babylonia infected with Shewanella abalone, but their intaking and moving ability was lost for a short time.[Conclusion] Although Shewanella algae and Shewanella abalone were the dominant bacteria in Babylonia suffered from proboscis edema, they were not the main pathogenic bacteria for proboscis edema. Shewanella algae had strong pathogenicity to mice, chicks and Babylonia, while Shewanella abalone showed no marked pathogenicity to those experimental animals in this study.

Acute toxicity of live and decomposing green alga Ulva (Enteromorpha) prolifera to abalone Haliotis discus hannai

From 2007 to 2009, large-scale blooms of green algae (the so-called "green tides") occurred every summer in the Yellow Sea, China. In June 2008, huge amounts of floating green algae accumulated along the coast of Qingdao and led to mass mortality of cultured abalone and sea cucumber. However, the mechanism for the mass mortality of cultured animals remains undetermined. This study examined the toxic effects of Ulva (Enteromorpha) prolifera, the causative species of green tides in the Yellow Sea during the last three years. The acute toxicity of fresh culture medium and decomposing algal effluent of U. prolifera to the cultured abalone Haliotis discus hannai were tested. It was found that both fresh culture medium and decomposing algal effluent had toxic effects to abalone, and decomposing algal effluent was more toxic than fresh culture medium. The acute toxicity of decomposing algal effluent could be attributed to the ammonia and sulfide presented in the effluent, as well as the hypoxia caused by the decomposition process.

Effects of temperature and salinity on survival, growth and DNA methylation of juvenile Pacific abalone, Haliotis discus hannai Ino

Temperature and salinity are two of the most potent abiotic factors influencing marine mollusks. In this study, we investigated the individual and combined effects of temperature and salinity on the survival and growth of juvenile Pacific abalone, Haliotis discus hannai Ino, and also examined the DNA methylation alteration that may underpin the phenotypic variation of abalone exposed to different rearing conditions. The single-factor data showed that the suitable ranges of temperature and salinity were 16-28℃ at a constant salinity of 32, and 24-40 at a constant temperature of 20℃, respectively. The two-factor data indicated that both survival and growth were significantly affected by temperature, salinity and their interaction. The optimal temperature-salinity combination for juveniles was 23-25℃ and 30-36. To explore environment-induced DNA methylation alteration, the methylation-sensitive amplified polymorphism （MSAP） technique was used to analyze the genomic methylation profiles of abalone reared in optimal and adverse conditions. Neither temperature nor salinity induced evident changes in the global methylation level, but 67 and 63 differentially methylated loci were identified in temperature and salinity treatments, respectively. The between-group eigen analysis also showed that both temperature and salinity could induce epigenetic differentiation in H. discus hannai Ino. The results of our study provide optimal rearing conditions for juvenile tt. discus hannai Ino, and represent the first step toward revealing the epigenetic regulatory mechanism of abalone in response to thermal and salt stresses.

Rheological Properties and Structural Changes in Different Sections of Boiled Abalone Meat

Changes in tissue structures, rheological properties of cross- and vertical section boiled abalone meat were studied in relation to boiling time. The adductor muscle of abalone Haliotis discus which was removed from the shell, was boiled for 1, 2, and 3 h, respectively. Then it was cut up and separated into cross- and vertical section meat. When observed by a light microscope and a scanning electron microscope, structural changes in the myofibrils were greatest in the cross section meat compared with the vertical section meat. When boiling time was increased from 1 h to 3 h, the instantaneous modulus E 0 and rupture strength of both section meat decreased gradually with increased boiling time, and no significant differences were observed between these two section meat for the same boiling time. When boiled for 1 h, the relaxation time of cross section meat was much longer than that of vertical section meat. There were no significant changes in the relaxation time of vertical section for different boiling time, but the relaxation time of cross section meat was reduced gradually with increasing boiling time. These results confirmed that the difference in rheological properties between the cross- and vertical section meat was mainly due to the denaturation level of myofibrils when heated for 1 h, as well as due to the changes in the amount of denatured proteins, and the manner in which the inner denatured protein components weve exchanged after boiling time was increased from 1 h to 3 h.

An AFLP Genetic Linkage Map of Pacific Abalone(Haliotis discus hannai)

A genetic linkage map of Pacific abalone(Haliotis discus hannai) was constructed using AFLP markers based on a two-way pseudo-testcross strategy in a full-sib family. With 33 primer combinations,a total of 455 markers(225 from the female parent and 230 from the male parent) segregated in a 1:1 ratio,corresponding to DNA polymorphism:heterozygous in one parent and null in the other. The female framework map consisted of 174 markers distributed in 18 linkage groups,equivalent to the H. discus hannai haploid chromosome number,and spanning a total length of 2031.4 cM,with an average interval of 13.0 cM between adjacent markers. The male framework map consisted of 195 markers mapped on 19 linkage groups,spanning a total length of 2273.4 cM,with an average spacing of 12.9 cM between adjacent markers. The estimated coverage for the framework linkage maps was 81.2% for the female and 82.1% for the male,on the basis of two estimates of genome length. Fifty-two markers(11.4%) remained unlinked. The level of segregation distortion observed in this cross was 20.4%. These linkage maps will serve as a starting point for linkage studies in the Pacific abalone with potential application for marker-assisted selection in breeding programs.

The Relationships between Rheological Properties and Structural Changes of Chilled Abalone Meat

The quantitative correlation between rheological properties and structural characteristic values of chilled abalone meat was studied. Structural changes were observed, and these values were enumerated using image processing and analysis technique. Structural changes in the myofibrils and collagen fibrils were the greatest in chilling for 24 h. After chilling for 48h, similar structures of vertical and cross sections were observed. For chilling from 0h to 72h, the instantaneous modulus E0 of the both section meat decreases gradually with time, but no significant differences were observed after chilling for 48h. The relaxation time and viscosity of both sections attained the same values for the same chilling time, but increased gradually with increasing chilling time. Meanwhile, a negative correlation between the structural characteristic values (Dm, Am, Rvm), and rheological properties (E1, τ1 , η1) clearly exists. Some logarithmic expressions have been obtained for these negative correlations. These results suggest that the difference in rheological properties between the cross and vertical sections was mainly due to the structural changes of myofibrils and collagen fibrils, and rheological properties are influenced quantitatively by the structural characteristic values for chilling from 0h to 72h.

Structural Changes and Rheological Properties of Dry Abalone Meat (Haliotis diversicolor) During the Process of Water Restoration

Changes in tissue structure, rheological property and water content of dry abalone meat in the process of water restora- tion were studied. The weight and volume of dry abalone meat increased with water restoration. When observed under a light micro- scope, structural change in myofibrils was obvious and a distinct network was found. When water restoration time increased from 24 h to 72 h, the instantaneous modulus E0 and viscosity η1 increased, whereas the rupture strength and relaxation time (τ1) were re- duced. There were no significant changes of rheological parameters (E0, η1, τ1, rupture strength) from 72 h to 96 h of water restoration. Therefore, the dry abalone meat was swollen enough at the time of 72 h. The rheological parameters were obviously influenced by the structural changes.

Diatom diet selectivity by early post-larval abalone Haliotis diversicolor supertexta under hatchery conditions

Benthic diatoms constitute the primary diet of abalone during their early stages of development. To evaluate the dietary preferences of early post-larval abalone, Haliotis diversicolor supertexta, we analyzed the gut contents of post-larvae that settled on diatom films. We compared the abundance and species diversity of diatom assemblages in the gut to those of the epiphytic diatom assemblages on the attachment films, and identified 40 benthic diatom species in the gut contents of post-larvae 12 to 24 d after settlement. The most abtmdant taxa in the gut contents were Navicula spp., Amphora copulate, and Amphora coffeaeformis. Navicula spp. accounted for 64.0% of the cell density. In the attachment films, we identified 110 diatom species belonging to 38 genera. Pennate diatoms were the dominant members including the species Amphiprora alata, Cocconeis placentula var. euglypta, Cylindrotheca closterium, Navicula sp. 2, and A. coffeaeformis. Nano-diatoms （〈20 pm in length） accounted for a considerable proportion of the total species number and cell density of the diatom assemblages in the gut contents and on the films. This suggests that nano-diatoms are important to the efficient production of abalone seed. The difference of the composition and abundance of diatoms between in the guts and on the biofilms suggests that early post-larval grazing was selective. An early post-larval abalone preferred nano-diatoms and the genera Navicula and Amphora during the month after settlement.

Determination of Content of Amino Acid in Abalone by Precolumn Derivatization and its Nutritional Value Evaluation

[Objectives] This study was conducted to optimize the determination conditions of amino acids from abalone. [Methods] The sample was treated by acid hydrolysis method and subjected to 2,4-2 nitro fluorobenzene column derivatization. The amino acid content in abalone was determined by HLPC,and the nutritional value of the amino acids was evaluated with egg protein model put forward by Institute of Nutrition and Food Hygiene,Chinese Academy of Preventive Medicine. [Results] Abalone contains full amino acids. According to the FAO/WHO ideal,it is a high-quality protein source and suitable for supplement of protein source for human body. [Conclusions]The experimental method has simple operation and could achieve a good effect with wide linear range and correlation coefficient over 0. 999 8,and the obtained results are satisfactory.

Development and Characterization of Microsatellite Markers for the Pacific Abalone (Haliotis discus) via EST Database Mining

The EST database of the Pacific abalone (Haliotis discus) was mined for developing microsatellite markers. A total of 1476 EST sequences were registered in GenBank when data mining was performed. Fifty sequences (approximately 3.4%) were found to contain one or more microsatellites. Based on the length and GC content of the flanking regions, cluster analysis and BLASTN, 13 microsatellite-containing ESTs were selected for PCR primer design. The results showed that 10 out of 13 primer pairs could amplify scorable PCR products and showed polymorphism. The number of alleles ranged from 2 to 13 and the values of Ho and He varied from 0.1222 to 0.8611 and 0.2449 to 0.9311, respectively. No significant linkage disequilibrium (LD) between any pairs of these loci was found, and 6 of 10 loci conformed to the Hardy-Weinberg equilibrium (HWE). These EST-SSRs are therefore potential tools for studies of intraspecies variation and hybrid identification.

Effects of Waterborne Cu and Cd on Anti-oxidative Response, Lipid Peroxidation and Heavy Metals Accumulation in Abalone Haliotis discus hannai Ino

The aim of this study was to compare the effects of waterbome copper （Cu） and cadmium （Cd） on survival, anti-oxida- tive response, lipid peroxidation and metal accumulation in abalone Haliotis discus hannai. Experimental animals （initial weight： 7.49 g±0.01 g） were exposed to graded concentrations of waterborne Cu （0.02, 0.04, 0.06, 0.08 mg L-1） or Cd （0.025, 0.05, 0.25, 0.5 mgL-1） for 28 days, respectively. Activities of the anti-oxidative enzymes （catalase, CAT; superoxide dismutase, SOD; glutathione peroxidases, GPx; glutathione S-transferase, GST）, contents of the reduced glutathione （GSH） and malondiadehyde （MDA） in the hepatopancreas, and metal accumulation in hepatopancreas and muscles were analyzed after 0, 1, 3, 6, 10, 15, 21, 28 days of metal exposure, respectively. Results showed that 0.04 mg L-1, 0.06 mg L-1 and 0.08 mgL-1 Cu caused 100% death of abalone on the 21st, 10th and 6th day, respectively. However, no dead abalone was found during the 28-day waterborne Cd exposure at all experimental concentrations. Generally, activities of SOD and GST in hepatopancreas under all Cu concentrations followed a decrease trend as the exposure time prolonged. However, these activities were firstly increased and then decreased to the control level and increased again during Cd exposure. Activities of CAT in all Cu exposure treatments were higher than those in the control. These activities were firstly increased and then decreased to the control level and increased again during Cd exposure. Contents of MDA in hepatopancreas in all Cu treatments significantly increased first and then decreased to the control level. However, the MDA contents in hepatopan- creas were not significantly changed during the 28-day Cd exposure. The metals accumulation in both hepatopancreas and muscles of abalone significantly increased with the increase of waterborne metals concentration and exposure time. These results indicated that H. discus hannai has a positive anti-oxidative defense against Cu or Cd. In conclusion, anti-oxidative mechanism in abalone to resist waterborne Cu did not follow the same pattern as that for waterborne Cd.

Characterization of genic microsatellite markers derived from expressed sequence tags in Pacific abalone (Haliotis discus hannai)

Simple sequence repeat (SSR) markers were developed from the expressed sequence tags (ESTs) of Pacific abalone (Haliotis discus hannai).Repeat motifs were found in 4.95% of the ESTs at a frequency of one repeat every 10.04 kb of EST sequences,after redundancy elimination.Seventeen polymorphic EST-SSRs were developed.The number of alleles per locus varied from 2-17,with an average of 6.8 alleles per locus.The expected and observed heterozygosities ranged from 0.159 to 0.928 and from 0.132 to 0.922,respectively.Twelve of the 17 loci (70.6%) were successfully amplified in H.diversicolor.Seventeen loci segregated in three families,with three showing the presence of null alleles (17.6%).The adequate level of variability and low frequency of null alleles observed in H.discus hannai,together with the high rate of transportability across Haliotis species,make this set of EST-SSR markers an important tool for comparative mapping,marker-assisted selection,and evolutionary studies,not only in the Pacific abalone,but also in related species.

Phylogenetic diversity of bacterial biofilms covering the settlement substrates of nona-porous abalones (Haliotis diversicolor supertexta)

The settlement substrates of nona-porous abalones （Haliotis diversicolor supertexta） are covered with biofilms in which several types of microorganisms coexist and interact. These microorganisms are usually important causes of juvenile abalone disease as well as organisms useful in promoting abalones’ adhesion. The bacterial community structure of the biofilms remains unclear. The aim of this research was to determine the genetic diversity and phylogenetic affiliation of the biofilm bacteria. Total DNA of bacteria in biofilms was extracted, and 16S rRNA gene clone library was constructed using the primers specific for the domain bacteria. Subsequently, 30 randomly selected positive clones were screened by PCR-restriction fragment length polymorphism （PCR-RFLP） analysis, and resulted in 15 different RFLP patterns. Sequences analysis of representatives from each unique RFLP type revealed high genetic diversity in the bacterial populations. These sequences fell into nine major lineages of the bacterial domains： α-, β-, γ-and δ-subdivisions of the Proteobacteria; Planctomycete, Actinobacteria, Firmicutes, V errucomicrobium spp., and CytophagaFlexibacter-Bacteroides spp. Phylogenetic analysis indicated that the dominant phylotypes were most closely related to environmental and clinical Burkholderia cepacia of the β-Proteobacteria, and Roseobacteria of the α-Proteobacteria.

Characterization of defensin gene from abalone Haliotis discus hannai and its deduced protein

Defensin is one of preserved ancient host defensive materials formed in biological evolution. As a regulator and effector molecule, it is very important in animals＇ acquired immune system. This paper reports the defensin gene from the mixed liver and kidney cDNA library of abalone Haliotis discus hannai Ino. Sequence analysis shows that the gene sequence of full-length cDNA encodes 42 mature peptides （including six Cys）, molecular weight of 4 323 Da, and pI of 8.02. Amino acid sequence homology analysis shows that the peptides are highly similar （70% in common） to other insects defensin. Because of a typical insect-defensin structural character of mature peptide in the secondary structure, the polypeptide named Haliotis discus defensin （hd-def）, a novel of antimicrobial peptides, belongs to insects defensin subfamily. The RT-PCR result of Haliotis discus defensin shows that the gene can be expressed only in the hepatopancreas by Gram-negative and positive bacteria stimulation, which is ascribed to inducible expression. Therefore, it is revealed that the Haliotis discus defensin gene expression was related to the antibacterial infection of Haliotis discus hannai Ino.

Effects of Dietary Carbohydrates Sources on Lipids Compositions in Abalone, Haliotis discus hannai Ino

A study was conducted to evaluate the effects of dietary carbohydrates on triglyceride, cholesterol and fatty acid concentrations in abalone, Haliotis discus hannai Ino. Six semi-purified diets with different carbohydrates （dextrin, heat-treated wheat starch, wheat starch, corn starch, tapioca starch and potato starch, respectively）, all containing a carbohydrate level of 33.5%, were fed to abalone （initial shell length： 29.98mm±0.09mm; initial weight. 3.42g±0.02g） for 24 weeks in a recirculation system. The results indicate that serum triglyceride concentrations were significantly （P〈0.05） higher in the abalone fed with dextrin, heat-treated wheat starch and wheat starch than those fed with corn starch, and serum cholesterol concentrations were significantly （P〈0.05） higher in the abalone fed with dextrin, heat-treated wheat starch than those fed with corn starch. Fatty acid C20：4n-6 in the foot mus- cles were significantly （P〈0.05） lower in the abalone fed with dextrin than those fed with wheat starch, corn starch, tapioca starch and potato starch. Fatty acid C20：4n-6 in hepatopancreas was significantly （P〈0.05） lower in abalone fed with heat-treated wheat starch than those fed with corn starch, tapioca starch and potato starch. Fatty acid C22：6n-3 in the foot muscles were significantly （P 〈 0.05） lower in the abalone fed with dextrin and beat-treated wheat starch than those fed with wheat starch and potato starch.

Effects of vitamin C deficiency or excess on growth performance, anti-oxidative response and fatty acid composition of juvenile abalone Haliotis discus hannai Ino

A 240-day feeding trial was conducted to investigate the effects of dietary vitamin C on growth performance,anti-oxidative response,and fatty acid composition of juvenile abalone Haliotis discus hannai Ino(initial body weight:0.93±0.00 g).Three semi-purified experimental diets were formulated containing 0.00,94.52,and 9649.58 mg/kg of vitamin C supplied as L-ascorbyl-2-monophosphate.The results show that there was no significant difference in weight gain ratio,daily increment in shell length,and survival rate among the three treatments.Adding dietary vitamin C(9649.58 mg/kg)significantly increased the activities of superoxide dismutase(SOD),glutathine peroxidase(GPX),glitathione-S-transferase(GST),glutathione reductase(GR),alkaline phosphatase(AKP),and lysozyme in viscera(P<0.05).In muscle,activities of phenoloxidase,catalase,SOD,GST,GR,and AKP were increased in the treatment with 9649.58 mg/kg of dietary vitamin C(P<0.05).The highest concentrations of ascorbic acid in viscera and muscle were found in the group with 9649.58 mg/kg of dietary vitamin C(P<0.05).The contents of crude protein and crude lipid in the soft body were significantly increased in the 9649.58 mg/kg group(P<0.05).Dietary vitamin C supplementation significantly decreased the contents of saturated fatty acids(14:0,16:0,and 18:0),and increased the composition of 18:2n-6 and 22:6n-3 in the soft body of abalone(P<0.05).Therefore,although there were no significant effects on the growth performance,dietary vitamin C supplementation improved the anti-oxidation and immune responses,increased specific unsaturated fatty acid(i.e.,16:1,18:1n-7,18:1n-9,18:2n-6 and 22:6n-3),and decreased specific saturated fatty acid(i.e.,14:0,16:0 and 18:0)contents in the soft body of abalone.

Studies on the Technology for Mass Production of Abalone Breeding

Based on the traditional way of abalone breeding,the technological process of industrial abalone breeding suitable for the coastal areas of southern China has been developed.The process is simple and easy to handle,and results in a high yield of young abalones after being applied.

Harmful Ciliates and Dominant Genus in Culture Ponds of Young Abalone

By using the methods of qualitative and quantitative analysis, the species composition and dominant genus of harmful ciliates in young abalone culture ponds are studied. Totally 68 ,species of ciliates, belonging to 3 classes16 orders and 46 genus, are found. The dominant ciliate species are Aspidisca leptaspis, Vorticella sp. and Euplotes rariseta. During the young abalone cultivation, the numbers of young abalones increase first, and then show a trend of decreasing. The relationship between the density and composition of ciliates and the density of algae in the young abalone culture ponds is also discussed.