A preliminary genetic map of Zhikong scallop (Chlamys farreri Jones et Preston 1904)

Zhikong scallop (Chlamys farreri Jones et Preston 1904) is one of the most important aquaculture species in China. The development of a genetic linkage map would provide a powerful tool for the genetic improvement of this species. Amplified fragment length polymorphism (AFLP) is a PCR‐based technique that has proven to be powerful in genome fingerprinting and mapping, and population analysis. Genetic maps of C. farreri were constructed using AFLP markers and a full‐sib family with 60 progeny. A total of 503 segregating AFLP markers were obtained, with 472 following the Mendelian segregation ratio of 1:1 and 31 markers showing significant (P<0.05) segregation distortion. The male map contained 166 informative AFLP markers in 23 linkage groups covering 2468 cM. The average distance between markers was 14.9 cM. The female genetic map consisted of 198 markers in 25 linkage groups spanning 3130 cM with an average inter‐marker spacing of 15.8 cM. DNA polymorphisms that segregated in a 3:1 ratio as well as the AFLP markers that were heterozygous in both parents were included to construct combined linkage genetic map. Five shared linkage groups, ranging from 61.1 to 162.5 cM, were identified between the male and female maps, covering 431 cM. Amplified fragment length polymorphism markers appeared to be evenly distributed within the linkage groups. Although preliminary, these maps provide a starting point for the mapping of the functional genes and quantitative trait loci in C. farreri.     

Cloning and characterization of tryptophan 2,3-dioxygenase gene of Zhikong scallop Chlamys farreri (Jones and Preston 1904)

A Zhikong scallop (Chlamys farreri Jones and Preston 1904) tryptophan 2,3‐dioxygenase (TDO) gene fragment, down‐regulated by Vibrio anguillarum challenge, was isolated using mRNA differential display in our previous work. In this paper, the full‐length TDO gene was cloned by 5′‐RACE. Chlamys farreri TDO gene consists of 1292 nucleotides encoding an expected polypeptide of 383 amino acids with an estimated molecular weight of 44.8 kDa and an isoelectric point of 6.35. The deduced amino acid sequence is 54–61% homologous to TDOs from Caenorhabditis elegans, Mus musculus, Danio rerio, Homo sapiens and Drosophila melanogaster, and shares several histidine residues important for the enzyme function in other species. Chlamys farreri TDO is expressed in the mantle, gill, digestive gland, testis, adductor muscle and kidney. Immunohistochemical analysis showed that C. farreri TDO was located mainly in the cytoplasm of most cell types. The non‐specific distribution of C. farreri TDO suggests that it is involved in various cellular processes.     

Effects of temperature on non-specific immune parameters in two scallop species: Argopecten irradians (Lamarck 1819) and Chlamys farreri (Jones & Preston 1904)

The effect of chronic (2 weeks) temperature (19, 22, 25, 28 and 31°C) on certain non‐specific immune parameters of two species of scallops, Argopecten irradians (Lamarck 1819) and Chlamys farreri (Jones & Preston 1904) were studied. The survival ratio of the two species of scallops at 31°C was the lowest. Haemocyte concentration of C. farreri in the 19 and 22°C treatment groups was significantly lower than that of the 25 and 28°C treatment groups but significantly higher than that of the 31°C treatment group. With elevation of seawater temperature, the alkaline phosphatase (ALP) and acid phosphatase (ACP) activities in sera of two scallop species increased and reached the peak at 25°C and then decreased. However, the specific activity of ALP and ACP of the 31°C treatment group was the highest. The results demonstrated that temperature of seawater significantly affected immunity of scallops. The concept that the stress of high environmental temperature on C. farreri may be partially responsible for the mass mortality of the organism is also introduced.     

附着生物对栉孔扇贝生长及存活的影响

2003年6-10月附着生物附着盛期对桑沟湾扇贝养殖笼上附着生物的种类组成、消长规律、附着生物量的季节变化以及附着生物对扇贝生长存活的影响方面进行了调查研究。结果表明：8月份附着生物总生物量达到最高峰,9月份开始逐渐下降;倒笼次数对栉孔扇贝的生长有较大影响,以每2个月倒换网笼1次、高温季节不倒笼为最佳。     

栉孔扇贝生物沉积作用的研究

2001年3～9月,在自然养殖状态下对栉孔扇贝(Chlamys farreri)的生物沉积及其对物质输运的影响进行研究.结果表明,栉孔扇贝能加速海洋中颗粒物质的沉积,生物沉积率分别为小个体(壳长30～40 mm)72.31～109.85 mg·ind-1·day-1、中等个体(壳长50～60 mm)103.49～207.77 mg·ind-1·day-1和大个体(壳长60～70 mm)120.05～237.65 mg·ind-1·day-1.栉孔扇贝的生物沉积与其壳长呈正相关线性关系,与其干组织重呈正相关的指数关系,而单位重量的生物沉积则与壳长和干组织重分别为负相关的线性和幂指数关系.海水温度和环境中饵料数量是影响栉孔扇贝的生物沉积的重要因子.     

Study on the potential of suspended longline mariculture of the scallop Chlamys farreri in offshore areas

The growth and survival of the scallop Chlamys farreri exposed to different initial densities were evaluated in culture systems from inshore and offshore areas of Sungo Bay, China, from May 2007 to March 2008. Water quality data (e.g. temperature, salinity, current speed, chlorophyll a and total particulate material) were measured concurrently. The C. farreri reached the market size after 10 months of cultivation. Average survival rates for the same initial densities were lower in the inshore area than those in the offshore area. Survival rates also were inversely proportional to the initial densities. Scallops in offshore area with initial densities of 20 and 30 individuals per disc resulted in significantly greater shell heights, soft tissue weights and muscle weight compared with the other experimental groups. Tissue weight increased rapidly from May to September. However, scallops in offshore area had a second growth peak during October to November. Scallops in the offshore area had higher growth and survival rates, which may be attributed to high water current speed resulting in increased food supplies. Results indicate that the scallop C. farreri has an aquaculture potential in offshore cultivation systems.     

栉孔扇贝神经节结构的显微观察

采用甲苯胺蓝、嗜银和铅苏木素3种组织学染色方法分别对栉孔扇贝(Chlamys farreri)神经节进行显微观察。结果表明,甲苯胺蓝染色法效果最好,神经细胞和神经纤维均着色较深,与背景反差较大。神经干及神经索中无神经元胞体,仅有沿神经索方向分布的神经纤维。神经节包括:1对彼此分离的脑侧神经节、1对连接在一起的足神经节和左右2神经节完全愈合而成的脏神经节。各神经节均由表面的神经节被膜、周边的胞体层和中央纤维网构成,胞体层分层现象不明显,神经元胞体根据其大小大致可分为直径15-36μm的大型细胞和5-15μm的小型细胞,胞体发出的突起均进入中央神经纤维网。脑侧神经节胞体层明显分脑神经节(CG)和侧神经节(PG)2区,CG区中神经元胞体主要分布于前端和外侧面,而PG区中神经元胞体则主要分布于外侧面;大型细胞较多,小型细胞很少。足神经节表层神经元胞体无分区分布的现象;大型细胞较少,而小型细胞较多。脏神经节相对较复杂,神经元胞体数目多,胞体层分区明显,分为:2个前叶,1个后叶,2个侧叶和2个嗅检叶;前叶和后叶中大型细胞较多,小型细胞较少;侧叶和嗅检叶中仅有小型细胞,而无大型细胞。     

三倍体栉孔扇贝的生殖腺观察

在栉孔扇贝（Chlamys farreri)的繁殖季节，对人工诱导的三倍体栉孔扇贝的生殖腺进行了外观，组织学及电镜观察，结果表明，三本栉孔扇贝的生殖腺外观呈透明状，分不出雌雄，组织切片观察，三倍体雌性的生殖腺滤泡细胞数量少，为一大的空腔，沿滤泡壁排列着单层不连续的卵原细胞，呈不规则形态，另外有许多三倍体的生殖腺组织内含有大量的不定性细胞，可能是尚未分化的生殖细胞，分布在稀少的滤泡内及及其周围，未发现卵原细胞和精原细胞，无法根据滤泡的组织学牧场生区分雌雄，在检查的样品中未发现雄性个体，电镜下观察到了三倍体的精母细胞，细胞结构呈梭形，核开始收缩，细胞质内线粒体较发达，但与二倍体相比细胞及线粒体数量稀少，说明三倍体栉孔扇贝存在着雄性个体，生殖细胞增殖分化出精原细胞和少数初级精母细胞后即停止了发育。     

栉孔扇贝鳃和唇瓣过氧化物酶活性

采用组织化学、电镜细胞化学和分光光度技术对栉孔扇贝(Chlamys farreri)鳃和唇瓣内的过氧化物酶(EC1.11.1.7;POD)进行研究,以探讨扇贝的鳃和唇瓣在免疫防御中的作用。组织化学显示,鳃轴、鳃丝和唇瓣上皮细胞均呈POD强阳性,结缔组织呈弱阳性;病毒感染后酶活力逐步增强。电镜细胞化学定位表明,鳃和唇瓣上皮细胞内有数量和大小不等的POD强阳性颗粒,阳性颗粒多为圆形,直径为150-220 nm;次级溶酶体也呈POD强阳性;内质网和空泡膜等细胞内膜系统以及细胞表面的微绒毛和纤毛呈POD弱阳性。结缔组织中部分血细胞呈POD强阳性。鳃轴上皮细胞内POD高电子密度阳性颗粒较少,而低电子密度阳性颗粒较多;主鳃丝上皮细胞内POD阳性颗粒较少,普通鳃丝上皮细胞内POD高电子致密阳性颗粒和呈POD强阳性的次级溶酶体均较多;唇瓣上皮细胞内POD阳性颗粒较鳃的略少。生化测定显示,鳃轴的POD酶活力最高,鳃丝次之,唇瓣最低;病毒感染24 h后,各部分的酶活力均显著增强。栉孔扇贝鳃和唇瓣内大量的POD可在抵抗病原微生物感染方面发挥重要的作用。     

栉孔扇贝稚贝造血组织的研究

采用石蜡切片和单克隆抗体的免疫组化方法,研究栉孔扇贝稚贝血细胞的分布和造血组织的定位。两种方法所得结果一致：在稚贝的外套膜、鳃、消化盲囊、闭壳肌、肾等处观察到大量血细胞,心脏位于消化腺与闭壳肌之间,闭壳肌、消化盲囊、肾等附近有血窦,血管分布于消化腺、胃、肾、直肠等处,有三个血窦,闭壳肌附近有一膨大突出的囊状组织,外被一层致密的结缔组织薄膜,其内壁血细胞密布层叠,分裂增生,形态多样,腔内充满了基质,靠近闭壳肌一侧有管道相通,并向闭壳肌等器官不断输送成熟的血细胞,该组织结构特点类似血细胞生发中心,并随着扇贝的生长发育逐渐膨大增生,据其结构特点及内部血细胞的形态、分布及免疫特性可初步定为栉孔扇贝稚贝的造血组织。     

栉孔扇贝血淋巴中酯酶活性及其性质

采用紫外分光光度法测定栉孔扇贝（Chlamysfarreri）血淋巴中酯酶的活性，并研究底物、抑制剂、激活剂、温度、pH对酯酶活力的影响及酶的热稳定性。结果表明，栉孔扇贝血清与血细胞中均存在酯酶，且血细胞中的酶活力高于血清。血清与血细胞中酯酶的最适作用底物有所不同，血清中为α-醋酸萘酯，而血细胞中为α-丁酸萘酯。当以α-醋酸萘酯为底物时，血清中酯酶的最适温度为45℃，最适pH值为7．5；以α-丁酸萘酯为底物时，血清中酯酶的最适温度为40℃，最适pH值为8．0。以α-醋酸萘酯及α-丁酸萘酯为底物分别测定酶的热稳定时，酶活力变化相似，在20℃时活力最高，而后缓慢下降。栉孔扇贝血淋巴中酯酶属中温型中性酶类，低温下较稳定，底物专一性不强，血清中的酯酶可能来自血细胞，该酶在贝类的免疫防御中町发挥重要的作用。     

胶州湾栉孔扇贝大规模死亡的流行病学调查

2 0 0 0年 1月 - 12月 ,跟踪调查了胶州湾养殖栉孔扇贝大规模死亡情况 ,对养殖海域环境因子进行了测定 ,并对大规模死亡期间发病栉孔扇贝进行了病理学观察。调查结果显示 ,胶州湾养殖栉孔扇贝大规模死亡呈暴发性并集中在 7月下旬的高水温期。发病高峰期在 7月 2 1日至 7月 2 7日 ,最高时点患病率达 14 .5 % ,7月份的月死亡率为 5 8.0 %。至 8月 3日累积死亡率达 90 .0 % ,患病贝死亡率接近 10 0 %。发病期扇贝壳高平均为 5 .0± 0 .9cm。,养殖栉孔扇贝半数死亡时间在 7月 2 3日。通过对水环境因子、病原性生物因子的调查分析及病理学观察表明 ,栉孔扇贝的大规模死亡可能是由生物性因子 (病毒 )所致 ,而与环境中理化因子可能无直接的相关关系     

Foxl2基因在栉孔扇贝发育过程中的表达图式

Foxl2基因在哺乳动物中是雌性相关基因,参与卵巢发育和功能维持。本研究利用荧光定量PCR和原位杂交技术,对栉孔扇贝(Chlamys farreri)foxl2(Cf-foxl2)在发育过程中的表达图式进行了分析。研究发现Cf-foxl2在受精卵中呈低水平表达,随着发育进行表达量增高,D形幼虫期表达量最高,之后表达量迅速下降。原位杂交结果显示Cf-foxl2表达量在担轮幼虫之前在体内均匀分布;强阳性信号在担轮幼虫口凹附近呈对称分布;至D形幼虫期强阳性信号主要集中在内脏团和外套膜边缘处;之后信号消失,直到性别分化后在卵巢中再次出现。本实验结果暗示Cf-foxl2与卵巢发育相关,鉴于该基因在发育早期的持续性表达特征,暗示其可能参与栉孔扇贝的早期发育过程。     

栉孔扇贝的滤食率与同化率

自青岛近海扇贝养殖区取栉孔扇贝(Chlamys farreri)暂养2周,壳长达25.01～73.92 mm.实验前停食24 h,实验温度梯度为8、13、18、23、28℃,其间投喂不同密度的小球藻(Chlorella spp.),静态实验.结果显示,栉孔扇贝的滤食率与温度和体重成正比,且与体重呈幂函数关系.在实验的温度范围内栉孔扇贝滤食率为1.07～11.66 mg/(ind     

栉孔扇贝雄核发育二倍体的人工诱导

研究了利用6-二甲基氨基嘌呤(60μg/mL;6-DMAP)抑制第1卵裂诱导栉孔扇贝[Chlamys farreri(Jones et Pre-ston)]雄核发育二倍体的条件。雄核发育单倍体是将强度为2.8 mW/(cm2.s)的紫外线照射20 s的卵子与正常精子受精后得到的。6-DMAP不仅可以有效地抑制减数分裂,还可以有效地抑制有丝分裂;抑制第1卵裂的适宜起始时间为受精后80 min,诱导率为21.0%~22.6%。细胞学观察显示,6-DMAP抑制第1卵裂产生的雄核发育二倍体主要来源于第1卵裂中期的受精卵,由于阻止了染色体分离和原核移动,导致一个融合的二倍性雌性原核的形成;经紫外线照射的卵核在第1卵裂中期没能形成染色体,而是形成1个浓缩的染色质小体,没有参与第1卵裂后期的核分裂。尽管倍化率和D形幼虫发生率较低,但本研究证实了栉孔扇贝雄核发育二倍体人工诱导的可行性。[中国水产科学,2006,13(4):585-590]     

Characterization of 95 novel microsatellite markers for Zhikong scallop Chlamys farreri using FIASCO-colony hybridization and EST database mining

ABSTRACT:   In order to construct a simple sequence repeat (SSR)-based genetic linkage map and to promote molecular marker-assisted selection (MAS) in scallop breeding, the methods of Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO)-colony hybridization and expressed sequence tag (EST) database mining were modified and used to develop 95 novel microsatellite markers for Zhikong scallop. The SSR-enriched library constructed by the FIASCO method consisted of 830 clones, and 295 (35.5%) positive clones were identified after colony hybridization. One hundred and fifty clones were randomly sequenced and the results showed all clones contained at least one microsatellite. Of 91 primer pairs designed, 72 were amplified scorable polymerase chain reaction (PCR) products and 70 were polymorphic with the allele number range of 3–16 alleles/locus (average 7.0 alleles/locus). When EST database mining was performed, 66 microsatellites containing ESTs were identified from 3467 sequences deposited in GenBank. Based on cluster analysis of length and GC content of the flanking regions, 47 primer pairs were designed and 23 scorable EST SSRs were obtained. Compared with genomic SSRs developed in this study, EST SSRs showed lower genetic variability with an average of 4.2 alleles/locus. The results in the present study demonstrate that modified FIASCO-colony hybridization is an efficient and low-cost method for the isolation of large numbers of microsatellite markers for scallop species.     

异源精子诱导栉孔扇贝雌核发育二倍体适宜参数的探索

采用照射强度为1500μw/cm2的紫外线对长牡蛎(Crassostrea gigas)精子照射60s,将处理后的精子分别与长牡蛎和栉孔扇贝(Chlamys farreri)的卵子进行受精试验,受精后11.5h用浓度为60mg/L的6-DMAP对栉孔扇贝受精卵分别持续处理15、20、25、30min,运用热滴片法对各实验组进行倍性检测。结果表明,长牡蛎同源单倍体组中单倍体率为75.6%;栉孔扇贝异源单倍体组中单倍体率为14.7%,最佳药物持续处理时间为20min,其二倍体率为24.1%;异源诱导栉孔扇贝单倍体组和二倍体组中胚胎均可以发育到D形幼虫期,其单倍体组中的D形幼虫率为5.0%,20min药物加倍二倍体组D形幼虫率最高为6.1%。本实验结果为异源精子诱导栉孔扇贝雌核发育提供了诱导参数依据。     

麻痹性贝类毒素在栉孔扇贝体内的代谢轮廓

将栉孔扇贝(Chlamys farreri)直接暴露于产毒藻,通过比较各组织器官中毒素的蓄积及代谢转化特征,研究麻痹性贝类毒素(Paralytic Shellfish Toxins,PSTs)在栉孔扇贝体内危害形成的过程。结果显示,分布于中国的一株塔玛亚历山大藻(Alexandrium tamarense,AT5-3株),其生长及产毒性状稳定,产毒能力强,主要成分为Gonyautoxins-1-4(GTX14),单细胞产毒能力为7.95 fmol/cell;栉孔扇贝对该产毒藻具有较强摄食能力及PSTs蓄积能力,总体蓄积效率达到84.4%,最高蓄积浓度为1903μg STX eq./kg,不同组织蓄积能力由高到低依次为内脏团、性腺、外套膜、闭壳肌;内脏团对PSTs的代谢能力也最强,是该毒素在栉孔扇贝体内蓄积代谢的靶器官;此外,栉孔扇贝对PSTs表现出较强的生物转化能力,主要转化途径为:N-磺酰胺甲酰基类毒素(N-Sulfocarbamoylgonyautoxin-2,C1)→膝沟藻毒素(Gonyautxins-2,GTX2)/脱氨甲酰基类毒素(Decarbamoylgonyautoxins-2,dc GTX2),N-磺酰胺甲酰基类毒素(N-Sulfocarbamoylgonyautoxin-3,C2)→膝沟藻毒素(Gonyautxins-3,GTX3)。本研究中,栉孔扇贝对PSTs总体呈现出迅速蓄积和缓慢代谢的特点,同时,GTX14和NEO等高毒成分所占比例较高,造成扇贝中PSTs毒性和风险性均显著升高。本研究有助于科学评估PSTs危害的形成机制,为系统监控我国PSTs风险性提供科学支撑。     

紫外辐射对栉孔扇贝精子遗传失活及形态结构的影响

报道了紫外辐射对栉孔扇贝精子遗传失活及形态结构的影响。采用光照强度为800μW/cm2·s的紫外线辐射40～50s的栉孔扇贝精子,与正常的卵子受精可以得到雌核发育的单倍体胚胎。随着辐射时间的增加,受精率明显下降,且受精卵极少发育到D形幼虫。扫描电镜观察发现,辐射后部分精子的顶体膜破裂,顶体丝伸出,辐射时间越长顶体受到的破坏越严重,证明紫外辐射可以诱导精子的顶体反应;精子的鞭毛在紫外辐射过程中也会受到破坏,随着辐射时间的增加鞭毛脱落的比例也逐渐增加。推测紫外辐射导致精子结构的破坏是受精率下降的原因之一。