栉孔扇贝血细胞的形态和功能研究

栉孔扇贝Chlamys farreri血细胞可分为三大类:非颗粒细胞(约34.75%)、颗粒细胞(约61.25%)和桑葚胚样细胞.非颗粒细胞可进一步分为透明细胞和成血细胞;颗粒细胞可分为嗜中性、嗜碱性和嗜酸性颗粒细胞.颗粒细胞能够吞噬酵母菌,其中嗜中性颗粒细胞吞噬能力最强.     

华贵栉孔扇贝♀×栉孔扇贝♂远缘杂交的受精细胞学观察

用HOECHST33258对华贵栉孔扇贝(Chlamys nobilis)♀×栉孔扇贝(Chlamys farreri)♂的受精卵进行染色,在荧光显微镜下观察其受精细胞学过程。观察表明:栉孔扇贝的精子能够使华贵栉孔扇贝卵子受精。精子入卵后呈一圆形亮点,体积稍有膨大成球形;授精后成熟卵母细胞释放出第一极体和第二极体后,精核解凝、稀疏、泡状化,形成雄性原核(male pronucleus);雌性原核(female pronucleus)在完成两次成熟分裂之后,染色质去浓缩,扩散膨大;雌雄原核相互靠拢,当雌雄原核膨胀到最大程度时,发生融合,形成合子。受精卵能够正常发育,完成第一次卵裂,卵裂时细胞核分离未观察到异常现象。与华贵栉孔扇贝和栉孔扇贝种内交配对照组相比,异源受精卵的细胞学过程明显滞后,同时其发生过程具明显的不同步现象。实验中还观察到少数的雌核发育现象。     

栉孔扇贝消化系统γ-氨基丁酸的免疫组织化学定位

采用抗生物素-生物素-过氧化物酶法(ABC法)的免疫组织化学技术,对γ-氨基丁酸(GABA)在栉孔扇贝(Chlamys farreri)消化系统各组织器官中的分布进行了定位研究。结果表明,栉孔扇贝的唇瓣、口唇、肠道的上皮细胞均呈GABA阳性反应,阳性反应物质呈颗粒状分布且多集中于上皮细胞游离端;胃上皮中未见GABA阳性反应;肝胰腺的上皮细胞及部分结缔组织中呈现GABA阳性反应,其中部分上皮细胞中的阳性反应物质呈团块状分布。GABA在栉孔扇贝消化系统除胃以外的各器官均有分布,推测其可能参与消化功能的调节作用。     

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。     

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。     

栉孔扇贝(♀)×虾夷扇贝( ♂ )受精细胞学观察

采用Bouin氏液固定、石蜡包埋、切片 ,用苏木精 伊红染色 ,在光学显微镜下观察栉孔扇贝 (♀ )与虾夷扇贝 (♂ )杂交的受精细胞学过程。尽管栉孔扇贝与虾夷扇贝同科不同属 ,但它们的杂交仍具有正常的受精细胞学程序。栉孔扇贝卵子处于第一次成熟分裂中期时接受虾夷扇贝的精子入卵 ,精卵混合后 6min精子入卵 ;8～ 1 0min精核略微膨胀 ;2 5～ 3 0min排出第一极体 ;1h左右 ,雌雄原核同时形成 ;1小时 3 0分钟左右 ,雌雄原核融合 ;2h左右开始卵裂。杂交过程中的精子入卵行为较本交迟缓 ,但杂交后代仍能正常发育     

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% .     

人工诱导栉孔扇贝雌核发育后代的微卫星标记分析

用紫外线灭活栉孔扇贝精子和6-DMAP处理受精卵抑制第二极体,获得A、B、C 3组雌核发育栉孔扇贝。采用筛选获得的5对多态性微卫星引物对3个家系雌核发育组30个个体、双亲及对照组40个个体进行了遗传变异分析。结果显示：3个家系雌核发育组后代中均有部分个体出现父本基因,表明精子遗传物质失活不彻底,雌核发育组中存在正常受精个体;在具多态性的5个基因座位上均发生了基因-着丝点之间的重组,重组率在26.7%—55.0%之间,表明通过抑制第二极体排出获得的栉孔扇贝雌核发育后代在个体和群体水平上具有一定的基因杂合;3个家系后代的平均基因纯合率为59.1%,而其对照组家系为2.5%,雌核发育使基因的纯合率提高了56.6%。研究表明雌核发育是促进基因纯合的一个有效途径,微卫星标记技术是贝类雌核发育鉴定和遗传分析的有效方法。     

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。     

Polymorphic microsatellite markers in the Zhikong scallop Chlamys farreri

The colony hybridization method was presented for isolating microsatellite markers in Zhikong scallop, Chlamys farreri. We designed primers to amplify 26 unique microsatellites. Fourteen primer pairs amplified clearly and were polymorphic. Based on characterization with 60 unrelated individuals, the number of alleles ranges from two to 13, and the expected heterozygosity ranges from 0.22 to 0.91. These markers have the potential for genetic studies in population structure and intraspecific variation.     

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．     

栉孔扇贝血细胞吞噬和包囊化作用实验方法的改进

为了建立一种快速、准确观察血细胞吞噬和包囊化作用的实验方法,通过抽取栉孔扇贝(Chlamysfarreri)血淋巴,与杆菌或茶花花粉作用30 min,制片,吖啶橙染色,用荧光显微镜观察吞噬或包囊化现象。结果表明,在荧光显微镜下可以明显看到扇贝血细胞呈现绿色,杆菌和茶花花粉呈现红色,两者颜色反差大,易于观察和计数。是研究血细胞吞噬和包囊化作用一种效果更好的实验方法。     

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.     

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     

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.     

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.