Isolation and characterization of 150 novel microsatellite markers for Zhikong scallop (Chlamys farreri)

We isolated and characterized 150 novel microsatellite markers of Zhikong scallop (Chlamys farreri) from three simple sequence repeat‐enriched libraries constructed with (GA)₁₅ and (CA)₁₅. The polymorphism was assessed with 48 individuals, and the result showed the number of allele ranged from two to 30, with an average of 8.4 alleles/locus. The values of observed and expected heterozygosities ranged from 0.0791 to 0.9878 and from 0 to 1.0000, respectively. Sixty‐five loci showed significant departure from Hardy–Weinberg equilibrium, and 14 locus pairs displayed linkage disequilibrium. These markers are therefore potentially useful for conservation studies, population structure assessment, ecological analyses and linkage map construction.     

Ten polymorphic dinucleotide microsatellite markers of the noble scallop Chlamys nobilis

The primers flanking 22 microsatellites isolated from a genomic library enriched for (CA)_n and (GA)_n were designed in the noble scallop Chlamys nobilis. Ten primer pairs provided clear and polymorphic amplification products. Based on characterization with 48 individuals, the number of alleles ranged from three to six. The values of observed heterozygosity and expected heterozygosity varied from 0 to 0.88 and from 0.29 to 0.76, respectively. No significant linkage disequilibrium between pairs of loci was found and six of 10 loci conformed to the Hardy–Weinberg equilibrium. These markers are therefore potentially useful for studies of the population structure of the species.     

Construction of microsatellite-based linkage maps and identification of size-related quantitative trait loci for Zhikong scallop (Chlamys farreri)

We constructed the microsatellite-based linkage maps using 318 markers typed in two F₁ outbred families of Zhikong scallop ( Chlamys farreri ). The results showed an extremely high proportion (56.2%) of non-amplifying null alleles and a high ratio (30%) of segregation distortion. By aligning different individual-based linkage maps, 19 linkage groups were identified, which are consistent with the haploid chromosome number of Zhikong scallop. The integrated linkage map contains 154 markers covering 1561.8 cM with an average intermarker spacing of 12.3 cM and 77.0% of genome coverage. We found that the heterogeneity in recombination rate was not determined by sexes but by different individuals on 18 linkage regions. The phenotypic marker of general shell colour was placed on LG4, which was flanked by microsatellite markers CFLD064 and CFBD055 . Four size-related traits including shell length (SL), shell width (SW), shell height (SH) and gross weight (GW) were analysed to identify the putative quantitative trait loci (QTL). Under the half-sib model, using dam as common parent, three, two, two and one QTL affecting SL, SW, SH and GW exceeded the genome-wide thresholds respectively. While using sir as common parent, a larger number of QTL were detected for these four traits: four, five, three and two for SL, SW, SH and GW respectively. The single QTL explained 3.7–19.2% of the phenotypic variation. The linkage map and the QTL associated with economic traits will provide useful information for marker-assisted selection of Zhikong scallop.     

栉孔扇贝生理生态学特征的实验研究

基于近年发展起来的生物沉积法,在烟台四十里湾养殖海区采用室内大型流水系统对栉孔扇贝的滤水率、吸收率和生态效率等生理生态学参数进行了测定,并探讨了它们与养殖密度与食物可获得性的关系．栉孔扇贝滤水率(平均3．65L·ind^-1·h^-1)与扇贝放养密度和饵料浓度无显著关系,而栉孔扇贝摄食率随放养密度的升高而降低,与POM呈正相关．扇贝对食物的吸收效率较高(平均75．9％),这与低饵料浓度行关．扇贝氨基酸泄漏所损失的能量高于排氨的能量损失．扇贝的生长余力(SFG)、总生长效率(尺：)和净生长效率(尺z)均与饵料浓度呈正相关．扇贝对氮的总生态效率(平均9．9％)高于对碳(平均5．9％)和磷(平均4．1％)．在氟的预算中,如果仅考虑NH4^ —N的排泄,而忽视其它形态氟的排泄,将会产生较大偏差(平均约20％)．贝壳不管在能量预算还是在元素预算中部不应该被忽视．在沿岸养殖海域,栉孔扇贝通过大量的滤水摄食、生长、排泄、生物沉积等对生态系统的能量流动和物质循环产生影响．     

栉孔扇贝血液细胞的免疫功能

利用光镜、扫描电镜和透射电镜技术对栉孔扇贝(Chlamysferreri)血细胞与细胞免疫功能相关的几个因素进行了初步研究。对血细胞的数量和不同功能细胞的比例研究结果表明,健康血淋巴中血细胞的平均密度为3.03±0.11×107cell/ml,其中颗粒细胞占42.6%,透明细胞占57.4%;病贝血淋巴中血细胞的平均密度为2.78±0.34×107cell/ml,其中颗粒细胞占40.2%,透明细胞占59.8%。扫描电镜观察表明,血细胞的表面结构主要有表面光滑型,表面松果型和表面褶皱阿米巴型3类。透射电镜观察表明,颗粒细胞吞噬外源性颗粒(Ⅰ型颗粒)通过溶酶体(Ⅱ型颗粒)进行降解。并观察到同心片层结构出现在吞噬泡的降解过程中。利用APIZYM试剂盒对栉孔扇贝血细胞及血清中的19种酶进行检测,结果在血清中检测到了13种酶,在血细胞中检测到10种酶,健康血淋巴中酶的含量高于病贝。对血细胞吞噬活性的研究结果表明,血细胞对大肠杆菌和对类立克次体(RLO)的吞噬率分别为25.4%和21.7%。颗粒细胞的吞噬活性(30%-40%)远远大于透明细胞(4.8%-14%)。环境胁迫对血细胞吞噬活性的影响的研究结果表明,病原菌感染和温度、盐度等环境胁迫因素对血细胞的吞噬活性均有不同程度的影响,其中高温因素影响较大,但未发现贝龄有显著影响     

桑沟湾栉孔扇贝生物沉积的现场测定

运用沉积物捕集器于桑沟湾对栉孔扇贝 (Chlamysfarreri)的生物沉积进行了现场测定 ,以评价贝类养殖对近岸生态环境的影响。结果表明 :桑沟湾栉孔扇贝具有相当高的生物沉积速率。壳高 68 1～77 9mm、软体干重 2 75～ 3 91g的栉孔扇贝 ,其生物沉积速率为 0 93～ 6 97g ind·d,平均 3 99g ind·d ;而壳高 40 8～ 67 4mm、软体干重 0 5 9～ 1 85g的栉孔扇贝生物沉积速率的变化范围为 0 5 2～ 6 42g ind·d,平均为 2 3 8g ind·d ;另外 ,壳高为 2 5 8～ 2 8 3mm、软体干重 0 1 2～ 0 1 7g的栉孔扇贝 ,其生物沉积速率在2 0 0 2年的 1 1和 1 2月以及 2 0 0 3年的 1月分别为 0 74、1 1 1和 0 1 5g ind·d。在桑沟湾 ,影响栉孔扇贝生物沉积的主要因素包括水温、悬浮颗粒物、扇贝个体大小和年龄。高密度、大规模的近岸浅海贝类养殖所产生的大量生物沉积物可能会对海区的物理、化学和生物环境产生影响。     

FISH Mapping and Identification of Zhikong Scallop (Chlamys farreri) Chromosomes

Chromosome identification is the first step in genomic research of a species, but it remains a challenge in scallops. In the present study, fluorescence in situ hybridization (FISH) mapping of 19 fosmid clones was attempted and used for chromosome identification in Zhikong scallop (Chlamys farreri Jones et Preston, 1904). Data showed that 10 clones were successfully mapped, including 7 without and 3 with C ₀ t-1 DNA. Among them, 2 represented multiple signals and made no contribution to chromosome identification. Karyotypic analysis and cohybridization indicated that the remaining 8 clones realized the identification of 8 chromosomes. All 10 clones were sequenced at both ends, which could be developed as sequence-tagged sites and used for the unification of the cytological and genetic linkage maps. This study shows that fosmid clones can benefit chromosome identification and will undoubtedly be useful for cytogenetic research in Zhikong scallop.     

一株形态特殊的细菌对栉孔扇贝致病性的初步研究

为研究分离到的一株形态特殊的细菌对栉孔扇贝(Chlamys farreri)致病性,采用测定病原液蛋白含量的方法,对病原相对定量。用含200～300μg／ml蛋白的病原液进行了不同浓度梯度、同一温度和相同浓度梯度、不同温度对栉孔扇贝的致病作用的测试,结果表明：扇贝人工感染后潜伏期3～7d,死亡高峰期为5～10d,符合一般病原感染的规律。病死贝的一般病理变化与养殖海区自然发病扇贝的病理变化一致。23、26℃下该病原体对栉孔扇贝具有强致病作用。可以确定该细菌为造成栉孔扇贝大规模发病死亡的主要病原之一。     

Polymorphic microsatellite loci for population studies of the Japanese scallop,Patinopecten yessoensis

Fifteen microsatellite loci were developed to examine the population structure of Japanese scallop, Patinopecten yessoensis. These markers were tested in samples from two geographically distant populations: the Sakhalin Island (Russia) and Dalian City (China). The mean numbers of alleles, observed and expected heterozygosities between the two populations were summarized by locus, but none of them show significant divergence between the two populations. Most loci conformed to Hardy–Weinberg expectation with the exception of loci HLJX‐06, HLJX‐12, HLJX‐13 and HLJX‐28, which had heterozygote deficits.     

cDNA cloning and mRNA expression of heat shock protein 90 gene in the haemocytes of Zhikong scallop Chlamys farreri

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. The full-length cDNA of Zhikong scallop Chlamys farreri HSP90 (designated CfHSP90) was cloned by EST and rapid RACE techniques. It was of 2710 bp, including an open reading frame (ORF) of 2181 bp encoding a polypeptide of 726 amino acids with all the five HSP90 family signatures. BLAST analysis revealed that the CfHSP90 gene shared high similarity with other known HSP90 genes. Fluorescent real-time quantitative RT-PCR was used to examine the expression pattern of CfHSP90 mRNA in haemocytes of scallops exposed to Cd²⁺, Pb²⁺ and Cu²⁺ for 10 and 20 days, respectively. All the three heavy metals could induce CfHSP90 expression. There was a clear dose-dependent expression pattern of CfHSP90 after heavy metals exposure for 10 days or 20 days. Different concentrations of the same metal resulted in different effects on CfHSP90 expression. The results indicated that CfHSP90 responded to various heavy metal stresses with a dose-dependent expression pattern as well as exposure time effect, and could be used as a molecular biomarker in a heavy metal polluted environment.     

栉孔扇贝急性病毒性坏死症病毒单克隆抗体的制备及ELISA检测

利用蔗糖密度梯度离心技术从自然发病的栉孔扇贝 (Chlamysfarrreri)组织分离纯化急性病毒性坏死症(Acutevirusnecrobioticdisease,AVND)病毒 ,并以此为抗原免疫Balb c小鼠。将免疫小鼠的脾细胞与NS_1骨髓瘤细胞进行融合 ,最终筛选出 4株能稳定传代并分泌该病毒特异性单克隆抗体 (MonoclonalAntibody ,MAb)的杂交瘤细胞系。应用胶体金标记免疫电镜技术在超微水平上对这 4株MAbs进行测定表明 ,它们对AVND病毒均具有高度的特异性 ,并且所识别的特异性位点均位于病毒粒子囊膜的纤突上。应用该单克隆抗体对不同养殖季节的一龄贝进行间接ELISA检测发现 ,7月中旬至 7月底病毒感染率与感染强度均处于当年的最高峰 ,与这一时期栉孔扇贝所表现出的最高死亡率完全吻合     

Characterization of novel microsatellite markers from the Yesso scallop Mizuhopecten yessoensis

Twelve polymorphic and informative microsatellite markers were isolated and characterized for the Yesso scallop, Mizuhopecten yessoensis. We characterized these loci by genotyping 48 individuals; the number of alleles ranged from two to eight, and the values of observed heterozygosity and expected heterozygosity varied from 0 to 0.8333 and from 0.2546 to 0.8231, respectively. No significant linkage disequilibrium between pairs of loci was found and three loci showed significant heterozygote deficiency from the Hardy–Weinberg equilibrium. These markers are potential for studies of the population structure, individual or hybrid identification of the species.     

Immunohistological detection of leucine-enkephalin and influence of leucine-enkephalin on the nitric oxide synthase activity of the scallop Chlamys farreri

The study was designed to determine whether leucine-enkephalin (L-ENK) was present on the nerve cells of the scallop Chlamys farreri. Furthermore, the constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) activity was investigated after different doses of L-ENK were added into the haemolymph of C. farreri. Some nerve cells immunoreactive to anti-leucine-enkephalin sera were observed in the cerebral ganglia and pedal ganglia. Intracellular and extracellular cNOS and iNOS activity of the haemolymph was induced with increasing concentration of L-ENK. The highest of the extracellular iNOS and cNOS activity was 0.84 ± 0.02 (U) and 1.30 ± 0.07 (U), respectively. The highest of the intracellular iNOS and cNOS activity was 1.51 ± 0.13 (U) and 2.11 ± 0.13 (U), respectively. Both the intracellular and extracellular iNOS and cNOS activity was highest when the concentration of L-ENK was 5 µg mL^?1. Higher or lower concentrations of L-ENK did not significantly induce the cNOS and iNOS activity. The data suggests an involvement of opioid peptides in the regulation and improvement of the immune processes of C. farreri.     

栉孔扇贝耗氧率和排氨率的研究

1999年 4～ 6月 ,采用室内实验生态学方法对栉孔扇贝的耗氧率和排氨率进行了研究 .结果表明 ,在适宜的温度范围内 ,栉孔扇贝的耗氧率和排氨率均与温度成正比 ,而与体重呈负相关关系 .在实验室温度 (8～ 2 8℃ )条件下 ,栉孔扇贝的耗氧率为 0 .48～ 9.0 9mg·g-1·h-1,排氨率为 0 .0 5～ 1 0 1mg·g-1·h-1.其中耗氧率在 2 3℃时达到最高值 ,2 8℃时开始下降 ,而排氨率则呈持续升高趋势 .栉孔扇贝的日常代谢明显高于标准代谢 ,耗氧率和排氨率平均值分别提高约 35 .8%和 75 .9% .     

Catecholaminergic responses to environmental stress in the hemolymph of Zhikong scallop Chlamys farreri

Catecholamines have long been known to play essential roles in several physiological processes in invertebrates. In this study, enzyme immunoassays were used to examine the levels of catecholamines (including adrenaline (AD), noradrenaline (NA) and dopamine (DA)) in the hemolymph of the scallop, Chlamys farreri, in responses to selected environmental stress. These stressors include high temperature (acute transfer from 17 to 28 degrees C), low salinity (acute transfer from salinity 31 to 20 psu) and air exposure, which are commonly encountered in aquaculture practices or in their natural habitat. The results showed that both AD and NA concentrations increased significantly in response to all the stressors. The levels of these catecholamines peaked 72 hr after acute temperature or salinity stresses, and then decreased gradually. For scallops subjected to air exposure, the duration of AD concentration elevation depended on the exposure temperature with a higher air temperature resulting in a shorter time to return to control level. In contrast, the NA responses were long lasting and no obvious decreases were observed after reaching the peak value. Interestingly, the DA concentration showed an inverse trend in response to stresses compared with AD or NA. These results suggested that the catecholaminergic responses in bivalve hemolymph will provide useful references in predicting the success of aquaculture practices and monitoring the environmental stresses.     

虾夷扇贝的多态性微卫星座位

虾夷扇贝(Patinopectenyessoensis),为冷水性贝类,是世界最重要的养殖经济贝类之一。原产于俄罗斯千岛群岛的南部水域,日本北海道及本洲北部。中国80年代初从日本引种,并开始养殖,目前已经在黄海北部形成规模化和产业化养殖,其中以大连长海养殖规模最大,近10年来创造出了数十亿     

栉孔扇贝感染急性病毒性坏死症病毒的组织病理学与免疫荧光检测

应用组织学和单克隆抗体免疫荧光技术(Immunofluorescence assay,IFA)对夏季养殖中后期大规模死亡(“急性病毒性坏死症”Acute virus necrobiotic disease,AVND)高峰期的患病栉孔扇贝(Chlamys,farrerl)进行了检测。组织学检测结果显示,患病扇贝的大多数器官(外套膜、鳃、胃、肾等)的上皮组织细胞可见显著的细胞肿胀、嗜碱性增强、排列紊乱、部分脱落以至完全坏死脱落等显著的组织病理学变化。作为对照,利用针对AVND病毒的特异性单克隆抗体所建立的免疫荧光原位检测技术对患病扇贝进行检测发现,上皮组织的病理变化与病毒感染之间具有一致的对应关系。这一结果表明,AVND病毒的感染可以对栉孔扇贝造成严重的病理性破坏。此为解释该病毒导致栉孔扇贝大规模死亡的机制提供了直接的组织病理学依据。     

Molecular cloning and characterization of a catalase gene from Zhikong scallop Chlamys farreri

Catalase is one of the central enzymes involved in scavenging the high level of reactive oxygen species (ROS) by degradation of hydrogen peroxide to oxygen and water. The full-length catalase cDNA of Zhikong scallop Chlamys farreri (denoted as CfCAT) was identified from hemocytes by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The nucleotide sequence of CfCAT cDNA consisted of 3146bp with a 5' UTR of 103bp, an unusually long 3' UTR of 1519bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1521bp encoding a polypeptide of 507 amino acids with predicted molecular weight of 57.5kDa. The deduced amino acid sequence of CfCAT has significant homology to catalases from animals, plants and bacteria. Several highly conserved motifs including the proximal heme-ligand signature sequence RLFSYNDTH, the proximal active site signature FNRERIPERVVHAKGGGA, and the three catalytic amino acid residues of His(72), Asn(145) and Tyr(355) were identified in the deduced amino acid sequence of CfCAT. The CfCAT was demonstrated to be a peroxisomal glycoprotein with two potential glycosylation sites and a peroxisome targeting signal of ANL that was consistent with human, mouse and rat catalases. The time-course expression of CfCAT in hemocytes was measured by quantitative real-time PCR. The expression of CfCAT increased gradually and reached the highest point at 12h post-Vibrio infection, then recovered to the original level at 24h. All these results indicate that CfCAT, a constitutive and inducible protein, is a member of the catalase family and is involved in the process against ROS in scallop.     

AFLP和RAPD标记技术在栉孔扇贝遗传多样性研究中的应用比较

AFLP和RAPD标记技术是近年来发展最快的基于PCR基础上的两种DNA标记技术,本文比较了两种标记技术在我国栉孔扇贝群体遗传多样性研究中的应用。共筛选20个RAPD引物和7个AFLP引物组合,检测到AFLP标记的有效等位基因数和平均多态信息量稍低于RAPD标记,但AFLP标记在每单位分析中扩增到的野生和养殖群体的多态性条带数(23．8,24．8)分别高于RAPD标记(5．6,5．6),AFLP多态性检测效率显著高于RAPD标记。AFLP和RAPD两种标记技术所揭示的野生种群与养殖群体间的近交系数、遗传距离两项指标均表明,我国栉孔扇贝养殖群体和野生种群之间尚未出现明显的遗传分化。研究结果表明：RAPD和AFLP这两种标记技术均可用于栉孔扇贝遗传多样性的分析,其分析结果是一致的。