皱纹盘鲍(Haliotis discus hannai)精子的超微结构

皱纹盘鲍（Ｈａｌｉｏｔｉｓｄｉｓｃｕｓｈａｎｎａｉ）的精子属于“原始型”，由顶体（２．２μｍ×１．０μｍ）、细胞核（３．１μｍ×０．８３μｍ）、中段（０．７４μｍ×１．４μｍ）和鞭毛（５２μｍ×０．２２μｍ）组成。顶体弹头状，由两部分组成：（１）顶体的前端是一呈卵形的高电子密度的部分；（２）顶体的后端为一呈翼状的低电子密度的部分。细胞核呈长柱状。在顶体凹陷和细胞核凹陷之间为顶体下腔，内有微丝束结构。中段包括５或６个线粒体，一对中心粒及一些泡状结构。鞭毛为典型的“９＋２”结构。

皱纹盘鲍(Haliotis discushannai Ino)胚胎RNA分离和cDNA文库构建

从皱纹盘鲍胚胎样本中分离RNA并构建了cDNA文库。分别收集孵化8、10和12h的皱纹盘鲍胚胎,用抽滤除菌的海水反复悬浮胚胎,将这3个发育阶段的胚胎样本等量混合后用TRIZOL试剂提取总RNA,将提取物用酚氯仿异戊醇再抽提2次,分离获得高质量的总RNA。从总RNA中分离mRNA,构建了皱纹盘鲍混合胚胎的cDNA文库,未扩增胚胎文库的滴度为5.0×106pfu/ml,重组率为94.12%,插入片段均大于400bp,70.6%克隆的插入片段分布在1000—1500bp之间,扩增后的文库滴度为3.06×109pfu/ml。

皱纹盘鲍(Haliotis discus hannai)肠道潜在益生菌的筛选及对幼鲍生长的影响

采用体外实验筛选益生菌结合16SrDNA序列分析法,从124株不具有溶血作用的皱纹盘鲍(Haliotis discus hannai)肠道细菌中筛选得到5株潜在益生菌并进行了分子鉴定,进一步对5株潜在益生菌进行了安全性实验及体内饲喂实验。结果表明,2株具有拮抗哈维弧菌和灿烂弧菌能力的潜在益生菌分别被鉴定为Shewanella sp.(WA64)和Shewanella sp.(WA65),一株产海藻酸酶和淀粉酶的潜在益生菌被鉴定为Vibrio sp.(WA51),产蛋白酶潜在益生菌被鉴定为Bacillus sp.(FA12),产琼脂酶潜在益生菌被鉴定为Tamlana sp.(FA86);安全性实验表明5株潜在益生菌在107cfu/ml下对皱纹盘鲍没有明显的毒害作用;通过体内饲喂实验发现,潜在益生菌WA64、WA65的复合作用能够显著提高幼鲍增重率和存活率(P<0.05),并在生产条件下能够明显降低幼鲍的死亡量。经抗生素敏感性实验,WA64菌株对15种抗生素均敏感或中度敏感,WA65菌株仅对庆大霉素和链霉素2种抗生素产生耐药。

皱纹盘鲍(Haliotis discus hannai Ino)数量性状的相关性与通径分析

为研究皱纹盘鲍各数量性状间的相关性及不同性状对体质量、软体部质量的影响，随机选取161只同一批次3-4龄的皱纹盘鲍，测量其壳长（SL）、壳宽（Sw）、壳厚（SH）、壳质量（SW）、足质量（FW）、软体部质量（MW）和体质量（BW）。计算性状间的相关系数，采用通径分析法计算不同性状对体质量、软体部质量的通径系数和决定系数，定量分析这些性状分别对体质量、软体部质量的影响效应。结果表明：所测各性状与体质量和软体部质量之间的相关系数均达到极显著水平（P＆lt;0.01）；在6个数量性状（壳长、壳宽、壳厚、壳质量、软体部质量、足质量）中软体部质量是影响体质量的主要因素；在体尺性状（壳长、壳宽、壳厚）中壳长对体质量影响最大；在体尺性状和体质量对软体部质量的影响中，体质量起主要作用。去除通径系数检验不显著的性状，利用逐步回归的方法，建立了6个数量性状对体质量的回归方程：BWq=-10.343＋0.255SL＋0.696SW＋0.951MW；体尺性状对体质量的回归方程：BWb=-69.930＋1.427SL＋0.728SH；体尺性状和体质量对软体部质量的回归方程：MB=4.430＋0.798BW-0.174Sw。研究结果为皱纹盘鲍的种质资源保护和选育工作提供一定的理论依据。

Genetic parameter estimates for growth traits at early stage of Pacific abalone,Haliotis discus hannai Ino

Heritability and genetic and phenotypic correlations were estimated for juvenile growth traits of Pacific abalone Haliotis discus hannai Ino. The estimates were calculated from shell length and shell width measurements on progeny resulting from 12 half-sib families and 36 full-sib families obtained using artificial fertilization of mating three females to each male. The measurements were taken at 10, 20 and 30 d after fertilization. It was found that heritability estimates based on sire component ranged from 0.23 to 0.36 for shell length and 0.21 to 0.32 for shell width. Heritability estimates from dam component were larger than those from sire component at three ages, indicating presence of maternal effects, non-additive genetic effects and common environmental effects. Phenotypic correlations were significant at three ages （P 〈 0.05）, with values of 0.92, 0.93 and 0.92, respectively. Genetic correlations from the paternal half-sib correlation analysis were highly positive at three ages, with values of 0.50, 0.78 and 0.81, respectively. The results suggest that selective breeding is an effective approach to improving growth traits of Pacific abalone stocks.

INDUCTION OF LARVAL SETTLEMENT AND METAMORPHOSIS OF HALIOTIS DISCUS HANNAI INO(GASTROPODA,MOLLUSCA)

Conspecific foot mucus,excessive[K^+] and gamma-aminobutyric acid (GABA) showed different metamorphosis-inductive effect on the veliger of Haliotis discus hannai.The inductive effect of excessive [K^+]and GABA was developmental stage-dependent and dose-dependent,while that of conspecific foot mu-cus was only developmental stage-dependent.At 20℃ the veliger larvae became competent within 4 daysafter fertilization H.discus hannai larvae showed gregarious settlement pattern on the conspecific foot mu-ous under the conditions of either presence or absence of KCI or GABA.The present studies showed that the effect of conspecific foot mucus on abalone larvae metarnorphosis could be dose-independent.

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.

Heterosis and combining ability:a diallel cross of three geographically isolated populations of Pacific abalone Haliotis discus hannai Ino

We conducted a complete diallel cross among three geographically isolated populations of Pacific abalone Haliotis discus hannai Ino to determine the heterosis and the combining ability of growth traits at the spat stage.The three populations were collected from Qingdao(Q) and Dalian(D) in China,and Miyagi(M) in Japan.We measured the shell length,shell width,and total weight.The magnitude of the general combining ability(GCA) variance was more pronounced than the specific combining ability(SCA) variance,which is evidenced by both the ratio of the genetic component in total variation and the GCA/SCA values.The component variances of GCA and SCA were significant for all three traits(P<0.05),indicating the importance of additive and non-additive genetic effects in determining the expression of these traits.The reciprocal maternal effects(RE) were also significant for these traits(P<0.05).Our results suggest that population D was the best general combiner in breeding programs to improve growth traits.The DM cross had the highest heterosis values for all three traits.

Sequences Characterization of Microsatellite DNA Sequences in Pacific Abalone(Haliotis discus hannai)

The microsatellite-enriched library was constructed using magnetic bead hybridization selection method, and the mi-crosatellite DNA sequences were analyzed in Pacific abalone Haliotis discus hannai. Three hundred and fifty white colonies were screened using PCR-based technique, and 84 clones were identified to potentially contain microsatellite repeat motif. The 84 clones were sequenced, and 42 microsatellites and 4 minisatellites with a minimum of five repeats were found (13.1% of white colonies screened). Besides the motif of CA contained in the oligoprobe, we also found other 16 types of microsatellite repeats including a dinucleotide repeat, two tetranucleotide repeats, twelve pentanucleotide repeats and a hexanucleotide repeat. According to Weber (1990), the microsatellite sequences obtained could be categorized structurally into perfect repeats (73.3%), imperfect repeats (13.3%), and compound repeats (13.4%). Among the microsatellite repeats, relatively short arrays (< 20 repeats) were most abundant, accounting for 75.0%. The largest length of microsatellites was 48 repeats, and the average number of repeats was 13.4. The data on the composition and length distribution of microsatellites obtained in the present study can be useful for choosing the repeat motifs for microsatellite isolation in other abalone species.

Cloning and Characterization of an Abalone(Haliotis discus hannai)Actin Gene

An actin encoding gene was cloned by using RT-PCR, 3’ RACE and 5’ RACE from abalone Haliotis discus hannai. The full length of the gene is 1532 base pairs, which contains a long 3’ untranslated region of 307 base pairs and 79 base pairs of 5’ untranslated sequence. The open reading frame encodes 376 amino acid residues. Sequence comparison with those of human and other mollusks showed high conservation among species at amino acid level. The identities was 96%, 97% and 96% respectively compared with Aplysia californica, Biomphalaria glabrata and Homo sapience β-actin. It is also indicated that this actin is more similar to the human cytoplasmic actin (β-actin) than to human muscle actin.

Acclimation-dependent expression of heat shock protein 70 in Pacific abalone (Haliotis discus hannai Ino) and its acute response to thermal exposure

Heat shock protein 70 （Hsp70） is one important member of heat shock protein （Hsp） family that is responsible for various stresses, especially thermal stress. Here we examined the response of Hsp70 gene to both chronic and acute thermal exposure in Pacific abalone （Haliotis discus hannai Ino）. For the chronic exposure, abalones were maintained at 8, 12, 20, and 30∩ for four months and their mRNA levels were measured. The highest mRNA level of Hsp70 gene relative to actin gene was detected in the 30~C-acclimated group, followed by the 8~C-acclimated group and then the 12~C- and 20~C-acclimated groups. After the long-term acclimation, gills from each of the above acclimation groups were dissected and exposed to different temperatures between 8~C and 38~C for 30 min. Hsp70 expression in gills acclimated to different temperatures responded differentially to the same temperature exposure. The incubation temperature that induced maximum Hsp70 mRNA expression was higher in the higher temperature acclimation groups than lower temperature groups. Pacific abalones could alter the expression pattern of Hsp70 gene according to environmental thermal conditions, through which they deal with the stress of thermal variations.

Experimental evaluation of heterobeltiosis and heterosis between two populations of Pacific abalone Haliotis discus hannai Ino

A2 × 2 factorial cross between two populations of Pacific abalone Haliotis discus hannai Ino, collected separately from Dalian （D） in China and Miyagi （M） in Japan, was conducted to compare performances in fertilization rate, hatching rate, metamorphosis rate and growth at days 20, 43, 160 and 330 between purebreds （DD and MM） and crossbreds （DM and MD） and investigate the magnitude of heterobeltiosis （better parent） and heterosis （mid-parent）. Heterobeltiosis and heterosis for all the traits analyzed were evidently different between crossbreds DM and MD. Heterobeltiosis in the crossbred DM varied among traits, with values of 2.5% for the fertilization rate, 2.2% for the hatching rate, - 1.9% for the metamorphosis rate and 7.4% for the growth at the day 330. The crossbred DM displayed positive heterotic values for fertilization rate （5.4%）, hatching rate （7.4%）, metamorphosis rate （7.6%） and growth （ 12.0% ） at the day 330. However, both heterobeltiosis and heterosis for all the traits in the crossbred MD were negative except those for the growth at days 20 and 43. The results indicate the importance of selecting superior hybrid varieties if the exploitation of hybrid vigor is considered in the Pacific abalone breeding program.

Effects of dietary menadione on the activity of antioxidant enzymes in abalone, Haliotis discus hannai Ino

A 240-day growth experiment in a re-circulating water system was conducted to investigate the effects of dietary menadione on the growth and antioxidant responses of abalone Haliotis discus hannai Ino. Triplicate groups of juvenile abalone (initial weight: 1.19 ± 0.01 g; shell length: 19.23 ± 0.01 mm) were fed to satiation with 3 semi-purified diets containing 0, 10, and 1 000 mg menadione sodium bisulfite (MSB)/kg, respectively. Results show that there were no significant differences in the rate of weight gain or in the daily increment in shell length of abalone among different treatments. Activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) in viscera were significantly decreased with dietary menadione. However, activities of these enzymes except for GPX in muscle were increased. Therefore, antioxidant responses of abalone were increased in muscle and decreased in viscera by dietary menadione.

Effects of dietary vitamin A on antioxidant responses of abalone Haliotis discus hannai Ino

A 240 d growth experiment was conducted in a re-circulated water system to investigate the effects of dietary vitamin A on growth and antioxidant responses of abalone Haliotis discus hannai Ino. Triplicate groups of juvenile abalone [ initial mass was （0.96±0. 02） g, shell length was （17.70±0.06） mm] were fed to satiation one of three send-purified diets containing 0, 1×10^3, 1×10^6 IU vitamin A per kilogram diet, respectively. Results showed that the daily increment in shell length （DISL） of abalone in the treatment with 1 × 10^3 IU vitamin A per kilogram diet was significantly higher than that with 0 or 1 × 10^6 IU vitamin A per kilogram supplementation （P 〈 0.05）. Vitamin A deficiency （0 IU/kg） significantly elevated the activities of superoxide dismutase （SOD）, glutathione peroxidase （GPX） and glutathione reductase （GR） in the viscera of abalone （P 〈 0.05）. In muscle, the effects of vitamin A deficiency on SOD and GPX activities were the same as those in viscera, however, the activity of GR significantly decreased （P 〈 0.05）. Vitamin A deficiency significantly decreased the ratio of CAT to SOD （eatalase/superoxide dismutase） in viscera （P 〈 0.05）. Nevertheless, it significantly decreased the ratio of GR to GPX in muscle （P 〈 0.05）. Compared with the supplement of 1 x 103 IU vitamin A per kilogram, excessive vitamin A （ 1 × 106 IU/kg） had no significant effects on the activities of CAT, SOD and glutathione-S-transferase （GST） （P 〉 0.05）, but significantly elevated GPX and GR activities in viscera （P 〈 0. 05）. In muscle, the activities of CAT, SOD, GPX, GST and GR were significantly decreased by the excessive dietary vitamin A supplement （P 〈 0.05）. Compared with the supplement of 1×10^3 IU vitamin A per kilogram, vitamin A-excessive had no significant effect on the value of ratio of CAT to SOD either in viscera or in muscle （ P 〉0.05 ）. The ratio of GR to GPX was significantly decreased in viscera, but significantly elevated in muscle in the vitamin A-excessive group （P 〈 0.05 ）. In conclusion, optimum supplement of vitamin A （ 1 × 103 IU/kg） was beneficial for abalone to maintain maximum growth and antioxidant system. Hypoor hyper-vitaminosis A would decrease growth and the efficiency of scavenging reactive oxygen species （ROS） of abalone.

AVAILABILITY OF PHOSPHORUS FROM SELECTED INORGANIC PHOSPHATES TO JUVENILE ABALONE,HALIOTIS DISCUS HANNAI INO.

The availability of phosphorus to juvenile abalone, Haliotis discus hannai from primary, secondary and tertiary calcium phosphate, primary sodium or potassium phosphate separately or in combination was determined in a 120-day feeding trial. Seven semi-purified diets were formulated to contain relatively constant dietary phosphorus, ranging from 0.78%-0.82%. The concentrations of dietary phosphorus were presumed to be slightly below the requirement for abalone. A brown alga, Laminaria japonica, was used as a control diet. Abalone juveniles of similar size (average weight 1.18 g; average shell length 18.74 mm) were distributed in a single-pass, flow-through system using a completely randomized design with eight treatments and three replicates each treatment. The survival ranged from 71.1% to 81.1%, and was not significantly (P>0.05) related to dietary treatment. However, the weight gain rate (41.72%-65.02%), daily increment in shell length (36.87-55.07 μm) and muscle RNA-DNA ratio (3.44-4.69) were significantly (P<0.05) affected by dietary treatment. Soft body alkaline phosphatase activity (10.9-19.8 U/g wet tissue) and carcass levels of lipid (7.71%-9.33%) and protein (46.68%-49.35%) were significantly (P<0.05) responsive to available phosphorus of the diets. Dietary treatment had significant effect (P<0.05) on concentrations of phosphorus in the whole body (WB) and soft body (SB). Apparent digestibility coefficients (45%-97%) of phosphorus were also significantly (P<0.05) different between dietary treatments. The results indicated that among these inorganic compounds, primary calcium, potassium and sodium phosphates separately or in combination could be utilized effectively by juvenile abalone as dietary phosphorus sources (availability ranging from 87%-97%). However, secondary and tertiary calcium phosphates were found to be low in availability, the values being 45% and 77%, respectively.

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.

Genetic variation of Chinese and Japanese wild Pacific abalone(Haliotis discus hannai)measured by microsatellite DNA markers

Population differentiation and relationships among three wild populations of the Pacific abalone Haliotis discus hannai collected from coastal seas around China and Japan were estimated using microsatellite DNA analysis. The results obtained with six microsatellite loci showed a high genetic diversity for China and Japan populations. The mean number of alleles per locus ranged from 11.7 to 23.0, and the average of observed and expected heterozygosity ranged from 0. 656 to 0. 721, and from 0. 721 to 0. 793, respectively. The observed genotype frequencies at each locus were mostly in agreement with Hardy - Weinberg expectations with five exceptions. Significant differences were detected between Chinese and Japanese H. discus hannai populations [ Weir and Cocker-ham＇ s fixation index（ Fst ） range ： 0. 020～ 0.023 ; Slatkin＇ s fixation index （Rst） range： 0. 016～ 0. 044 ], and no obvious difference was detected between the samples of Japanese H. discus hannm populations （ Fst = 0. 002 ; Rst = 0. 007）. The level of differentiation among populations is further evidenced by the nNeighbor-joining tree topology on which the Japanese samples were closely clustered, and the Chinese population formed a separate cluster. These results suggest that care should be taken in future management of different populations.

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.

Identification of normalization factors for quantitative realtime RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai

Quantitative real-time reverse transcription-polymerase chain reaction （qRT-PCR） is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Fibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues （digestive glands, foot muscle, gill, hemocyte, and mantle） were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-l-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.

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.