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.

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.

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.

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.

First steps on technological and genetic improvement of European abalone (<i>Haliotis tuberculata</i>) based on investigations in full-sib families

The European abalone Haliotis tuberculata is of economical importance in Europe, and recently in France where most of the consumed abalones remained wild animals fished on the coast. Recently, the creation of hatcheries allowed the production of cultured animals, and aquaculture is in progress. To optimize selective breeding programs, different studies were performed on adults and on their progenies. First, ten adult families, assumed to be bi-parental and produced in 2007 were analyzed. As these families were developed at the beginning of the hatchery production, the parentage of the individual necessitated to be tested. The progenies parentage assignment was done by using eight microsatellite DNA loci. In fine, the heritability was estimated from the analysis of variance of seven full-sib families which include from 23 to 27 progenies. Heritability estimate was the higher for length, width and weight (0.37, 0.29 and 0.40 respectively). A high correlation was also shown between these heritable traits which can be useful during animal breeding. Shell color traits were also analyzed, using image treatment procedure. Two traits were selected according to the global color of the shell: red color and hue. The distribution of these traits evidenced a polygenic genetic control of shell color and measure of heritability evidenced a high to medium value for red color (Cr) and Hue(H) (0.33 and 0.20 respectively). No correlation was shown between growth and this parameter. In a second time, juveniles families produced in 2010 were studied from parents issued of the first selection. A correlation between size and density at juvenile stage was established but a statistical analysis demonstrated, by using density as co-variable a significant effect of family on size. In these conditions, growth is highly heritable (0.74). This study is a first step toward the use of genetic markers for selection, but also a step toward the breeding improvement of European abalone.

The co-effect of microstructures and mucus on the adhesion of abalone from a mechanical perspective

Reliable and reversible adhesion underwater is challenging due to the water molecules and weak layers of contaminants at the contact interface,which requires to deepen the un-derstanding of wet adhesion of biological surfaces.Herein,the co-effect of microstruc-tures and mucus of abalone foot on wet adhesion is investigated from both experimental and theoretical perspectives.The morphologies,adhesion force and coefficient of friction indicate that the mucus in adhesion zone is crucial for successful attachment of abalone based on capillary forces and viscous forces,and the mucus in non-adhesion zone with lower adhesion force and friction coefficient may behave as a lubricant for the loco-motion.The theoretical calculation manifests that the microstructures may help abalone to form multiple liquid bridges with the secreted mucus,and significantly increase the wet adhesion force of abalone.These findings will bring profound views into the underlying mechanisms of biological surface adhesion.

哈维弧菌和鲍类疱疹病毒刺激对杂色鲍免疫相关因子的影响

以哈维弧菌(Vibrio harveyi)、鲍类疱疹病毒(Abalone herpesvirus,Ab HV)悬液对杂色鲍(Haliotis diversicolor)注射刺激,研究其血淋巴中可溶性总蛋白浓度,免疫相关酶超氧化物歧化酶(Superoxide Dismutase,SOD)、酸性磷酸酶(Acid Phosphatase,ACP)、碱性磷酸酶(Alkaline Phosphatase,AKP)活性的变化,杂色鲍外套膜、鳃、肝胰腺、腹足中血蓝蛋白(Hemocyanin,Hc)基因Hc1、Hc2相对表达量的变化以及感染后杂色鲍无细胞血浆的抑菌性。结果显示:(1)杂色鲍血淋巴中可溶性总蛋白浓度变化显著,呈现出先升高后降低的趋势。(2)无细胞血浆中的SOD、ACP、AKP活性与对照组相比变化显著,哈维弧菌注射组的SOD活性在感染后第6小时显著升高;Ab HV注射组SOD活性与对照组比较在注射后的各个时相均呈下降趋势,从注射后6 h各个时相的SOD活性与对照组相比均差异显著(P<0.05)。哈维弧菌注射组ACP活性在注射后的第48小时显著升高;Ab HV注射组ACP活性在注射后的第12小时显著升高。哈维弧菌注射组AKP活性在注射后48 h显著升高;AbHV注射组AKP活性在注射后第12小时显著升高。(3)杂色鲍外套膜、鳃、肝胰腺以及腹足中血蓝蛋白两个基因Hc1、Hc2的相对表达量均在注射后的不同时间点出现升高。(4)杂色鲍无细胞血淋巴对哈维弧菌、创伤弧菌和溶珊瑚弧菌这三种贝类病原均表现出了不同程度的抑菌性增强的作用。这些结果表明细菌和病毒的刺激引起了杂色鲍免疫相关因子的变化,使得血淋巴的抑菌性增强。本研究为今后进一步研究杂色鲍的非特异性免疫以及在生产实践中应用免疫技术防治病害提供参考。

Mechanical properties and structure of Haliotis discus hannai Ino and Hemifusus tuba conch shells:a comparative study

Haliotis discus hannai Ino （abalone shell） and Hemifusus tuba conch shell have been studied for the purpose to comparatively investigate the mechanisms by which nature designs composites. It is shown that both shells are composed of aragonite and a small amount of proteins while the conch shell shows finer microstructure but lower strength than aba- lone shell. It is also shown that the fresh shells exhibits better property than those after heat-treatments. It is therefore sup- posed that the size of inorganic substance is not a dominant factor to improve strength, while both proteins in shells and the microstructure of inorganic matter also play important roles.

The roles of thyroid hormone receptor and T3 in metamorphosis of Haliotis diversicolor

Thyroid hormone is a kind of important hormone which regulates metamorphosis. Its role is well described in amphibian metamorphosis. Thyroid hormones (T3 and T4) have also been demonstrated to play a role in metamorphosis of marine invertebrates. However, the mechanism of thyroid hormone in metamorphosis of marine invertebrates remains unknown. A homolog of vertebrate thyroid hormone receptor (TR) was cloned and identified in abalone Haliotis diversicolor and was named HdTR . The mRNA expressions of HdTR , thyroid peroxidase ( TPO ), thyroid peroxidase 1 ( TPO1 ), idothyronine deiodinase Ⅲ( IDⅢ) and integrin alpha-V ( ITGAV ) had significant diff erence in metamorphosis of H . diversicolor . Metamorphosis rate and mortality rate were significantly diff erent in HdTR RNAi experiment and T3 inducing experiment. In RNAi experiment, ITGAV and CCND1 (cyclin D1) expression of dsRNA HdTR exposing group were significantly lower than those of blank control and negative control. But CTNNB (catenin beta) expression of dsRNA HdTR exposing group was higher than that those of blank control and negative control. ERK (extracellular signal regulated kinases) and PI3K (phosphoinositide-3-kinase) had no significant diff erence in RNAi experiment. Moreover, ITGAV of 1 μmol/L T3 group was significantly lower than that of 0 μmol/L T3 group, PI3K expression of 10 μmol/L T3 group was higher than that of 0 μmol/L T3 group, and the other genes expression had no significant diff erence in T3 inducing experiment. The data of genes expression suggested that CCND1 might be an eff ector gene of TR genomic action, while CTNNB might be regulated by unliganded TR. CCND1 and CTNNB may be involved in cell proliferation of metamorphosis. T3 might regulate the expression level of PI3K via nongenomic way. These results shed light on the mechanism of thyroid hormone in abalone metamorphosis.

Detection of two pathogenic marine ciliates Ancistrum haliotis and A. crassum(Ciliophora: Scuticociliatia) by fluorescence in situ hybridization

The scuticociliatid ciliates Ancistrum haliotis and A.crassum are parasites that may cause high mortality in the cultured abalone Haliotis spp.and the bivalve Ruditapes philippinarum.Traditional identification with silver staining methods is hampered by their morphological similarities to closely related species and the complicated procedures of morphological analysis.We designed two SSU rRNA-targeted oligonucleotide probes labeled with a fluorochrome,and optimized the fluorescence in situ hybridization(FISH)protocols for identification of A.halioti and A.crassum,respectively.The assays resulted in a clear identification by strong fluorescence signals from the oligonucleotide probes.The method can be used for quick and accurate quantitative analysis of A.haliotis and A.crassum infections on host molluscs.

A Bioinspired Adhesive Sucker with Both Suction and Adhesion Mechanisms for Three-Dimensional Surfaces

There are significant advantages to investigate underwater attachments,which would be valuable in providing inspirations and design strategies for multi-functional surfaces and underwater robots.Here,an abalone-inspired sucker integrating an elastic body and a membrane structure is proposed and fabricated filled with rigid quartz particles to adjust the backing stiffness of the contact like abalone.The membrane is used to conform and contact surfaces well,the center area of which can be pulled in exposed to a negative pressure differential,to create a suction cavity.The pulling experiments indicate that the sucker can adhere to three-dimensional surfaces with both suction and adhesion mechanisms in both dry and liquid environments.The switching between soft/hard contact states leads to the change of adhesive strength over 30 times.Furthermore,we provide theoretical analysis on how the sucker work well in both dry and liquid environments.Finally,the developed sucker can easily lift up smooth planar objects and 3D objects,and can grip objects both smaller and larger than the size of the sucker,which have a difficulty for conventional suckers or friction-based grippers.The potential application of the sucker in flexible transfer robot is demonstrated on various surfaces and environments,paving the way for further bio-inspired adhesive designs for both dry and wet scenarios.