A centromeric satellite sequence in the Pacific oyster (Crassostrea gigas Thunberg) identified by fluorescence in situ hybridization

A highly repetitive satellite sequence was previously identified in the Pacific oyster Crassostrea gigas Thunberg. The sequence has 168 bp per unit, present in tandem repeats, and accounts for 1% to 4% of the genome. We studied the chromosomal location of this satellite sequence by fluorescence in situ hybridization (FISH), A probe was made by polymerase chain reaction and incorporation of digoxigenin-11-dUTP. Hybridization was detected with fluorescein-labeled antidigoxigenin antibodies. FISH signals were located at centromeric regions of 7 pairs of the Pacific oyster chromosomes. No interstitial site was found. Signals were strong and consistent on chromosomes 1, 2, 4, and 7, but weak or variable oil chromosomes 5, 8, and 10. No signal was observed on chromosomes 3, 6, and 9. Our results showed that this sequence is clearly a centromeric satellite, disputing its previous assignment to the telomeric and submetacentric regions of 2 chromosomes. No signal was detected in the American oyster (Crassostrea virginica Gmelin).

Chromosome inheritance in triploid Pacific oyster Crassostrea gigas thunberg

Reproduction and chromosome inheritance in triploid Pacific oyster (Crassostrea gigas Thunberg) were studied in diploid female x triploid male (DT) and reciprocal (TD) crosses. Relative fecundity of triploid females was 13.4% of normal diploids. Cumulative survival from fertilized eggs to spat stage was 0.007% for DT crosses and 0.314% for TD crosses. Chromosome number analysis was conducted on surviving progeny from DT and TD crosses at 1 and 4 years of age. At Year 1, oysters from DT crosses consisted of 15% diploids (2n = 20) and 85% aneuploids. In contrast, oysters from TD crosses consisted of 57.2% diploids, 30.9% triploids (3n = 30) and only 11.9% aneuploids, suggesting that triploid females produced more euploid gametes and viable progeny than triploid males. Viable aneuploid chromosome numbers included 2n + 1, 2n + 2, 2n + 3, 3n - 2 and 3n - 1. There was little change over time in the overall frequency of diploids, triploids and aneuploids. Among aneuploids, oysters with 2n + 3 and 3n-2 chromosomes were observed at Year 1, but absent at Year 4. Triploid progeny were significantly larger than diploids by 79% in whole body weight and 98% in meat weight at 4 years of age. Aneuploids were significantly smaller than normal diploids. This study suggests that triploid Pacific oyster is not completely sterile and cannot offer complete containment of cultured populations.

Seasonal variation in metabolism of cultured Pacific oyster, Crassostrea gigas, in Sanggou Bay, China

Rates of respiration and excretion of the Pacific oyster, Crassostrea gigas, were measured seasonally from June 2002 to July 2003 under ambient conditions of food, water temperature, pH, and salinity in Sanggou Bay, an important mariculture coast in north China. The aim of this study is to obtain fundamental data for further establishing an energy budget model and assessing the carrying capacity for cultivation of C. gigas in north China. Oysters were collected monthly or bimonthly from the integrated culture areas of bivalve and kelp in the bay. Oxygen consumption and ammonium and phosphorus excretion rates were measured, and ratios of O/N and NIP were calculated. One-way ANOVA was applied to determine differences among these parameters that act as a function of seasonal variation. All the physiological parameters yielded highly significant variations with season (P<0.01) The rate of respiration varied seasonally, with the highest oxygen consumption rate in July and the lowest rate in January, ranging from 0.07 to 2.13 mg O-2 h(-1) g(-1) dry tissue weight (DW). Maximum and minimum ammonium excretion rates were recorded in August and January, respectively, ranging from 0.51 to 5.40 mu mol NH4-N h(-1) g(-1) DW. Rates of phosphorus excretion varied from 0.11 (in January) to 0.64 (in July) mu mol PO4-P h(-1) g(-1) DW. The O/N and N/P ratios changed from 9.2 (in January) to 59.8 (in July) and from 4.6 (in January) to 10.9 (in August), respectively. For each season, the allometric relationship between the physiological response (e.g., rate of oxygen consumption, ammonium and phosphorus excretion) and DW of the animal was estimated using the formula: Y=a x DWb. (C) 2005 Elsevier B.V. All rights reserved.

Inheritance of 15 microsatellites in the Pacific oyster Crassostrea gigas: segregation and null allele identification for linkage analysis

Microsatellites were screened in a backcross family of the Pacific oyster, Crassostrea gigas. Fifteen microsatellite loci were distinguishable and polymorphic with 6 types of allele-combinations. Null alleles were detected in 46.7% of loci, accounting for 11.7% of the total alleles. Four loci did not segregate in Mendelian Ratios. Three linkage groups were identified among 7 of the 15 segregating loci. Fluorescence-based automated capillary electrophoresis (ABI 310 Genetic Analyzer) that used to detect the microsatellite loci, has been proved a fast, precise, and reliable method in microsatellite genotyping.

AFLP-based genetic linkage maps of the Pacific oyster Crassostrea gigas Thunberg

Amplified fragment length polymorphisms (AFLPs) were used for genome mapping in the Pacific Oyster Crassostrea gigas Thunberg. Seventeen selected primer combinations produced 1106 peaks, of which 384 (34.7%) were polymorphic in a backcross family. Among the polymorphic markers, 349 were segregating through either the female or the male parent. Chi-square analysis indicated that 255 (73.1%) of the markers segregated in a Mendelian ratio, and 94 (26.9%) showed significant (P < 0.05) segregation distortion. Separate genetic linkage maps were constructed for the female and male parents. The female framework map consisted of 119 markers in 11 linkage groups, spanning 1030.7 cM, with an average interval of 9.5 cM per marker. The male map contained 96 markers in 10 linkage groups, covering 758.4 cM, with 8.8 cM per marker. The estimated genome length of the Pacific oyster was 1258 cM for the female and 933 cM for the male, and the observed coverage was 82.0% for the female map and 81.3% for the male map. Most distorted markers were deficient for homozygotes and closely linked to each other on the genetic map, suggesting the presence of major recessive deleterious genes in the Pacific oyster.

Chromosome segregation in fertilized eggs from triploid Pacific oysters, Crassostrea gigas (Thunberg), following inhibition of polar body 1

Chromosome segregation in fertilized eggs from triploid Pacific oysters, following inhibition of the first polar body (PB1), was studied with acetic orcein staining techniques. To block the release of PB1, fertilized eggs were treated with 0.5 mg/l of cytochalasin B (CB). Four types of segregation were observed, namely, ''tripolar segregation'' (54.5%), ''united bipolar segregation'' (12%), ''separated bipolar segregation'' (2.5%), and ''incomplete united bipolar segregation'' (4%). The remaining 23% could not be classified because of chromosome disorganization, but appeared to be variants of the above. It seemed clear that the predominant pattern that gave rise to tetraploids was united bipolar segregation, although certain separated bipolar segregations might also lead to the formation of tetraploids. The sequential events of meioses observed in CB-treated eggs are described. The asynchrony of meiotic events and possible mechanisms for the various types of chromosome segregation are discussed.

Microstructure Observation And Mechanical Behavior Modeling For Limnetic Nacre

In the present research, microstructure of a kind of limnetic shell (Hyriopsis cumingii) is observed and measured by using the scanning electron microscopy, and mechanical behavior experiments of the shell nacre are carried out by using bending and tensile tests. The dependence of mechanical properties of the shell nacre on its microstructure is analyzed by using a modified shear-lag model, and the overall stress-strain relation is obtained. The experimental results reveal that the mechanical properties of shell nacre strongly depend on the water contents of the limnetic shell. Dry nacre shows a brittle behavior, whereas wetting nacre displays a strong ductility. Compared to the tensile test, the bending test overestimates the strength and underestimates the Young's modulus. The modified shear-lag model can characterize the deformation features of nacre effectively.

Differences in the rDNA-bearing chromosome divide the Asian-Pacific and Atlantic species of Crassostrea (Bivalvia, Mollusca)

Karyotype and chromosomal location of the major ribosomal RNA genes (rDNA) were studied using fluorescence in situ hybridization (FISH) in five species of Crassostrea: three Asian-Pacific species (C. gigas, C. plicatula, and C. ariakensis) and two Atlantic species (C. virginica and C. rhizophorae). FISH probes were made by PCR amplification of the intergenic transcribed spacer between the 18S and 5.8S rRNA genes, and labeled with digoxigenin-11-dUTP. All five species had a haploid number of 10 chromosomes. The Atlantic species had 1-2 submetacentric chromosomes, while the three Pacific species had none. FISH with metaphase chromosomes detected a single telomeric locus for rDNA in all five species without any variation. In all three Pacific species, rDNA was located on the long arm of Chromosome 10 (10q)-the smallest chromosome. In the two Atlantic species, rDNA was located on the short arm of Chromosome 2 (2p)-the second longest chromosome. A review of other studies reveals the same distribution of NOR sites (putative rDNA loci) in three other species: on 10q in C. sikamea and C. angulata from the Pacific Ocean and on 2p in C. gasar from the western Atlantic. All data support the conclusion that differences in size and shape of the rDNA-bearing chromosome represent a major divide between Asian-Pacific and Atlantic species of Crassostrea. This finding suggests that chromosomal divergence can occur under seemingly conserved karyotypes and may play a role in reproductive isolation and speciation.

The cDNA cloning and mRNA expression of cytoplasmic Cu, Zn superoxide dismutase (SOD) gene in scallop Chlamys farreri

Cu, Zn superoxide dismutases (SODs) are rnetalloenzymes that represent one important line of defence against reactive oxygen species (ROS). A cytoplasmic Cu. Zn SOD cDNA sequence was cloned from scallop Chlamys farreri by the homology-based cloning technique. The full-length cDNA of scallop cytoplasmic Cu, Zn SOD (designated CfSOD) was 1022 bp with a 459 bp open reading frame encoding a polypeptide of 153 amino acids. The predicted amino acid sequence of CfSOD shared high identity with cytoplasmic Cu. Zn SOD in molluscs, insects, mammals and other animals, such as cytoplasmic Cu, Zn SOD in oyster Crassostrea sostrea gigas (CAD42722), mosquito Aedes aegypti (ABF18094), and cow Bos taurus (XP_584414). A quantitative reverse transcriptase real-time PCR (qRT-PCR) assay was developed to assess the mRNA expression of CfSOD in different tissues and the temporal expression of CfSOD in scallop challenged with Listonella anguillarum, Micrococcus luteus and Candida lipolytica respectively. Higher-level mRNA expression of CfSOD was detected in the tissues of haemocytes, gill filaments and kidney. The expression of CfSOD dropped in the first 8-16 h and then recovered after challenge with L. anguillarum and M. litteus, but no change was induced by the C. lipolytica challenge. The results indicated that CfSOD was a constitutive and inducible acute-phase protein, and could play an important role in the immune responses against L. anguillarum and M. luteus infection. (C) 2007 Elsevier Ltd. All rights reserved.

Concurrence of cat and tet genes in multiple antibiotic-resistant bacteria isolated from a sea cucumber and sea urchin mariculture farm in China

A basic understanding of abundance and diversity of antibiotic-resistant microbes and their genetic determinants is necessary for finding a way to prevent and control the spread of antibiotic resistance. For this purpose, chloramphenicol and multiple antibiotic-resistant bacteria were screened from a mariculture farm in northern China. Both sea cucumber and sea urchin rearing ponds were populated with abundant antibiotic-resistant bacteria, especially marine vibrios. Sixty-five percent chloramphenicol-resistant isolates from sea cucumber harbored a cat gene, either cat IV or cat II, whereas 35% sea urchin isolates harbored a cat gene, actually cat II. The predominant resistance determinant cat IV gene mainly occurred in isolates related to Vibrio tasmaniensis or Pseudoalteromonas atlantica, and the cat II gene mainly occurred in Vibrio splendidus-like isolates. All the cat-positive isolates also harbored one or two of the tet genes, tet(D), tet(B), or tet(A). As no chloramphenicol-related antibiotic was ever used, coselection of the cat genes by other antibiotics, especially oxytetracycline, might be the cause of the high incidence of cat genes in the mariculture farm studied.

贝藻混养系统中贝类食物来源的定量分析

应用稳定碳、氮同位素比值法和脂肪酸标志分析法，较为系统地研究了贝藻混养系统中滤食性贝类的食物来源，评估了大型藻类对混养系统及滤食性贝类的物质贡献。主要研究结果如下： 1.综述了典型生态系统中大型藻类和滤食性贝类各自的生态学地位和作用，大型藻类与滤食性贝类不仅在水体营养盐方面存在互利关系，二者在物质循环与收支方面同样具有耦合性，大型藻类提供的颗粒态有机质可以为滤食性贝类提供饵料来源。 2.总结了稳定同位素比值法和脂肪酸标志法在海洋生态系统食物来源及食物网分析中的应用，并建立了两种方法的具体操作规程。 3.分析了栉孔扇贝Chlamys farreri和海带Laminaria japonica混养系统中海带碎屑形成及释放不同阶段的生态学特征，评估了碎屑对扇贝的饵料贡献。海带在6周内释放了自身约27％的碳；碎屑形成及释放过程中C:N比值显著下降，同时伴随着旺盛的细菌降解，碎屑中也发现有大量硅藻类和原生动物存在。稳定同位素分析证实海带碎屑是混养期间扇贝的主要食物来源。 4.查明了春季胶州湾潮间带自然分布的长牡蛎Crassostrea gigas、紫贻贝Mytilus galloprovincialis和湾内浅海筏式养殖栉孔扇贝的可能食物来源。湾内栉孔扇贝饵料组成中浮游硅藻类为最主要部分，同时混杂有陆源有机质和细菌类物质；潮间带自然生长的牡蛎和贻贝饵料组成中，浮游植物占86.2-89.0％，种类组成中除硅藻外还包括一定比例的金藻和甲藻类；潮间带繁盛的孔石莼Ulva pertusa藻床为两种贝类提供了8.7-11.0％的补充食物来源。 5.揭示了桑沟湾贝藻混养海区春、夏季栉孔扇贝饵料来源组成情况及其季节变化，评估了海带养殖区碎屑碳量季节变化及海带来源碳对扇贝组织碳的贡献。结果表明，湾内贝藻混养区碎屑碳量为75.52-265.19 μg l-1，其在水体总颗粒态有机碳中的比例为25.6-73.8％。海带来源碎屑碳对栉孔扇贝组织碳的贡献比例为14.1-42.8％，且与水体碎屑碳比例的季节变化存在极显著相关性(F=0.992, P=0.004)。5月份湾外海带养殖区水体碎屑碳量为110.12-144.71 μg l-1，显著高于湾内无海带区（75.52 μg l-1），湾外养殖的扇贝组织中海带来源碳比例为22.0-24.1％，显著高于湾内单养区扇贝（9.6％）。估算结果表明，桑沟湾每年收获的6967吨（总湿重）栉孔扇贝中，海带提供了约57.1吨碳，换算为海带干物质为219.6吨。脂肪酸标志分析结果表明，2月份至8月份硅藻类在扇贝饵料组成中比例逐渐下降，而细菌类比例逐渐升高。整个采样期间，EPA/DHA比例较低，说明扇贝饵料组成中可能包括高DHA含量的组分。

双壳贝类幼虫变态诱导及其机理研究

变态过程是双壳贝类由幼虫向成体转变的一个必不缺少的发育阶段。研究双壳贝类幼虫的变态过程及其机理，对于阐明它们的种群数量变动，促进重要经济双壳贝类增养殖的发展有重要的理论和实践意义。本论文除了用化学物质对几种双壳贝类(海湾扇贝、墨西哥湾扇贝和硬壳蛤)幼虫的变态进行诱导外，主要以激素和神经递质的作用方式为基础，通过直接测定双壳贝类(以海湾扇贝为代表)幼虫体内激素和神经递质、第二信使cAMP等生化物质含量的变化来研究双壳贝类幼虫变态过程中的信息传递途径，从分子生物学和神经生物学角度阐明双壳贝类幼虫变态机理。主要结果如下：1．通过参考国内外大量文献的基础上，较为系统地评述了近二十年来海洋无脊椎动物幼虫附着变态研究的一些进展情况，主要包括诱导因子、附着变态机理模型、人工诱导物的应用和延迟变态四个方面。到目前为止，人们已经发现了许多海洋无脊椎动物幼虫附着变态的诱导物质，主要分为天然诱导物和人工诱导物两大类，一些人工诱导物如GABA、肾上腺素和去甲肾上腺素已经在经济贝类苗种生产中得到应用。幼虫附着变态机理模型主要有长牡蛎(Crassostrea gigas)幼虫附着变态的双调控模型、红鲍(Haliotis rufescens)幼虫附着变态的上行调节模型以及多毛类Phragmatopoma california幼虫附着变态的脂肪酸调控模型。本论文还评述了海洋无脊椎动物幼虫发生延迟变态的原因以及延迟变态对海洋无脊椎动物造成的影响，并提出了解决的方法和今后研究的重点问题。2．在室内用氯化乙酰胆碱、ATP和CaCl_2 3种化学物质对海湾扇贝幼虫的变态进行了诱导实验。结果表明，虽然在个别浓度和处理时间氯化乙酰胆碱和ATP有诱导作用，但总体诱导效果不显著。而10×10~(-3)～40×10~(-3M的CaCl_2在处理12～24h后诱导效果较显著，其诱导效果对处理时间的依赖性较显著，在浓度为40×10~(-3)M和处理时间为24h时诱导效果最好，与对照组相比，变态率提高23.18％。3种诱导物对幼虫死亡率均有显著影响，并且死亡率对浓度和处理时间均有显著的依赖性，浓度越高，处理时间越长，死亡率越高。3．用KCl、肾上腺素、去甲肾上腺素和氯化胆碱进行了墨西哥湾扇贝(Argopectenirradians concentricus Say)幼虫变态的诱导作用实验。结果表明，KCl、肾上腺素、去甲肾上腺素和氯化胆碱对墨西哥湾扇贝幼虫变态均有显著诱导作用。KCl在处理时间为12h～48h范围内均有诱导作用；13.42×10~(-3)M和20.13×10~(-3)M的KCl诱导效果较好，变态率平均提高10％以上。1．O×10~(-6)M～50×10~(-6)M的肾上腺素在处理时间为lh～12h较适宜，此时变态率均提高10％以上。1.0×10~(-6)M～50×10~(-6)M的去甲肾上腺素在处理时间为1h～24h都较适宜，变态率平均均提高10％以上，最高可提高31．07％。0.01×10~(-4)M～1.O×10~(-4)M的氯化胆碱在处理时间为12h～48h时诱导效果均较好，它们之间的平均变态提高率并没有显著差别，均在12％～13％之间。10×10~(-4)M的氯化胆碱在处理时间为12h时诱导效果较明显，变态率可以提高19.14％，超过12h，变态率明显下降，100×10~(-4)M的氯化胆碱明显产生毒害作用，幼虫变态率均为零，而幼虫的死亡率均为100％。4．用KCl、肾上腺素、去甲肾上腺素、L-DOPA、5-羟色胺(5-hydroxytryptamine，Serotonin，5-HT)和GABA(γ-氨基丁酸)进行了不同浓度不同处理时间对硬壳蛤(Mercenaria mercenaria L.)幼虫变态诱导实验。结果表明，KCl、肾上腺素、去甲肾上腺素、L-DOPA和5-羟色胺对硬壳蛤幼虫的变态均有诱导作用，而GABA的诱导 作用不显著。KCl的最佳诱导浓度随处理时间不同而有所不同。当处理时间为1～24h时，KCl的最佳诱导浓度为33.56×10~(-3)M，此时幼虫变态率均提高24％以上，当处理时间为48h时，KCl的最佳诱导浓度为20.13～26.85×10~(-3)M，处理时间为72h时，最佳诱导浓度为13.42×10~(-3)M。肾上腺素和去甲肾上腺素的诱导作用与浓度和处理时间均有关。肾上腺素的最佳处理浓度为100×10~(-6)M，最佳处理时间均为8h，此时幼虫变态率提高最大，为36.97％。当去甲肾上腺素的诱导浓度为100×10~(-6)M，处理时间为8h～16h时，幼虫变态提高率较高，均大于18％，死亡提高率均低于30％，当去甲肾上腺索诱导浓度为500×10~(-6)M时，虽然在8h～16h的处理时间范围内，幼虫变态提高率也较高，均大于18％，但当处理时间超过8h，在16～48h范围内，幼 虫死亡提高率明显升高，均大于50％。L-DOPA的适宜诱导浓度为10×10~(-6)M～50×10~(-6)M，适宜处理时间为8～24h，此时幼虫变态率均提高30％以上，最高可提高79.43％。5-羟色胺的诱导作用较强，其适宜诱导浓度为100×10~(-6)M—1000×10~(-6)M，适宜处理时间为0.5～24h，此时幼虫变态率提高均在30％以上，当处理时间为8h时，最佳诱导浓度为1000×10~(-6)M，此时幼虫变态率提高57.5％，当处理时间为24h时，最佳诱导浓度为100×10~(-6)M，此时幼虫变态率提高69.29％。GABA的诱导作用较弱，最佳诱导浓度随处理时间的不同而有所不同。处理时间为24h和48h时，最佳诱导浓度为0.1×10~(-6)M；处理时间为0.5～16h时，最佳诱导浓度为100×10~(-6)M。5．KCl、肾上腺素、去甲肾上腺索、L-DOPA、5-羟色胺、GABA、茶碱和咖啡因8种诱导物对不同发育阶段海湾扇贝幼虫变态的诱导作用是不同的。13．42×10~(-3)M和20.13×10~(-3)M的KCl对第12天幼虫的变态有抑制作用，变态提高率为负值；之后当幼虫发育至第13和14天时，两浓度的KCl能够明显诱导幼虫变态，变态提高率均高于20％，而对于第16天的幼虫诱导作用有所减弱，变态提高率有所降低；26.85×10~(-3)M的KCl对第12和13天幼虫的变态均有抑制作用，变态提高率为负值，对第14和16天幼虫的变态却有明显的持续的诱导作用，变态提高率分别为22.98％和37.5％。神经递质肾上腺素、去甲肾上腺素、L-DOPA、5-羟色胺和GABA的诱导作用规律基本相似，即对第13天海湾扇贝幼虫的变态有明显的抑制作用，变态提高率均为负值，而对第14天幼虫的诱导作用较显著。茶碱和咖啡因作为影响细胞内cAMP的物质，它们的诱导作用规律与神经递质有所不同。它们对第13天海湾扇贝幼虫变态的诱导效果最好。6．测定了不同发育阶段及人工诱导后海湾扇贝幼虫体内去甲肾上腺素、多巴胺和5-羟色胺含量的变化规律。结果表明，海湾扇贝幼虫体内去甲肾上腺索含量在变态前和变态后没有明显变化，变态前为2352(pg／mg湿重)，变态后为2770(pg／mg湿重)。多巴胺和5-羟色胺含量在变态前随幼虫的发育而增加，变态前(第13天)急剧增加，第13天的幼虫比第12天的幼虫分别增加了2.8倍和5.7倍，变态后急剧下降，变态后幼苗比第13天的幼虫分别降低了25.1倍和16.4倍。海湾扇贝幼虫体内DA：NE比和5-HT：NE比在变态前和变态后变化比较剧烈。DA：NE比和5-HT：NE比在变态前(第13天)急剧增加，第13天的幼虫比第12天的幼虫增加了3.O倍(DA：NE比)和5.0倍(5-HT:NE比)；变态后急剧降低，变态后幼苗比第13天的幼虫降低了29.8倍(DA：NE比)和19.5倍(5-HT：NE比)。海湾扇贝幼虫经KCl和氯化钙诱导24h后，体内去甲肾上腺素、多巴胺和5-羟色胺以及DA：NE比和5-HT：NE比均有所降低。本实验的结果表明，多巴胺和5-羟色胺可能启动了海湾扇贝幼虫的变态过程。7．茶碱和咖啡因对墨西哥湾扇贝幼虫的变态均有明显诱导作用。它们的诱导作用均对浓度的依赖性较强，对处理时间的依赖性较弱。10×10~(-4)M的茶碱诱导效果最好，平均变态提高率达33％，其次为1.0×10~(-4)M和100×10~(-4)M的茶碱，平均变态提高率分别为23.15％和21.97％。处理时间对茶碱诱导效果影响不显著，在1～24h范围内，平均变态提高率在19.07～26.1％之间变动。10×10~(-4)M的咖啡因诱导效果最佳，4个处理时间的平均变态提高率为36.01％，其次为100×10~(-4)M，平均变态提高率为26.43％。处理时间对茶碱的诱导效果影响不大，在1～24h范围内，平均变态提高率在19.65～22.02％之间变动。8．采用直接测定cAMP的方法来研究cAMP是否参与了海湾扇贝幼虫的变态过程。结果表明，cAMP参与了海湾扇贝幼虫的变态过程。海湾扇贝幼虫体内cAMP含量随着发育阶段的不同而有所变化。在D形幼虫期最低，为73 pmol／(mg蛋白质)；当到达壳顶期幼虫时cAMP含量明显增加，比D形幼虫期提高了12.7倍。从壳顶期幼虫到眼点幼虫(100％，第13天)cAMP含量增加速度较慢，各发育阶段分别比前一发育阶段增加了0.4倍、0.3倍和0.2倍。但当幼虫变态后，体内cAMP含量又急剧增加，幼苗体内cAMP含量比眼点幼虫(100％，第13天)增加了6.1倍。当用KCl、肾上腺索和L-DOPA诱导后，幼虫体内cAMP含量明显增加，分别比对照组提高了7.8倍、1.5倍和10.7倍，说明cAMP参与了这3种诱导物诱导海湾扇贝幼虫变态的过程。9．在前面实验结果和参考有关文献的基础上，初步提出了以海湾扇贝为代表的双壳贝类幼虫变态机理模型：幼虫变态分为两个过程：启动过程和后续过程。当幼虫发育到一定阶段，在外界刺激因子的作用下，体内分泌多巴胺和5-羟色胺，多巴胺和5-羟色胺通过某种信号转导途径(如以DG和IP_3为第二信使)启动变态过程，变态过程启动后，又激活以cAMP为第二信使的信号转导途径(暂时称为后续过程)，两者共同完成了幼虫的变态过程。

南极普里兹湾大、中型浮游动物群落生态学研究

在南大洋生态系统中，桡足类既在初级生产和更高营养级之间起着承上启下的作用，也为大型浮游动物，主要是磷虾生物量年际变化和地区变化提供了很好的缓冲，磷虾存在时它是竞争者和捕食对象，磷虾缺乏时初级生产的转化主要由它承担。国际上对南极桡足类的研究在最近十年进展很快，尤其是生活史策略的研究取得了长足进步。我国对南极磷虾的研究已处于国际先进水平，但对桡足类等小型浮游动物来说却只是刚刚起步。本文利用中国第十三、十五次南极考察的浮游动物标本研了普里兹湾的游游动物群落结构和地理分布。随纬度的不同，普里兹湾存在三种群落类型，62－65°海域是以纽鳃樽为主的北部被囊群落，小型桡足类拟长腹剑水蚤（Oithona similis）和以哲水蚤（Paracalanus spp）在该区域密度最大，最大型的巨锚哲水蚤（Rhincalanus gigas）为该区所特有，并且只有62°S附近出现大量的无节幼体；65－67°S之间是尖角似哲水蚤（Calanoides acutus）和戈氏长腹水蚤（Metridia gerlachei）的分布高峰区，多毛类和毛颚类分布也较多，该地区由磷虾和桡足类交替控制；67°S以南至近岸为近岸群落，是晶磷虾（Euphausia crystallorophias）的分布区域，在我们的调查中由大量的磷虾幼体出现，无节幼体最大密度6000ind·m~(-2)，蚤状幼体达到6400ind·m~(-2)。划分北部和中部群落的指示种为纽鳃樽（Salpa thommpsoni）、巨锚哲水蚤和毛颚类；北部和近岸群落的指示种为巨锚哲水蚤、戈氏长腹水蚤和磷虾的蚤状幼体；中部群落和近岸群落之间的指示种是晶磷虾，磷虾的无节幼体和角突隆剑水蚤（Oncaea conifera）作为该区的指示种不是很明显。CHINARE－XV期间进行了桡足类优势种是产卵量培养实验，在调查其间尖角似哲水蚤和戈氏长腹水蚤分别有13.3和5只／雌的产卵量，尖角似哲水蚤表现为明显的双峰态种群结构，早期幼体占据绝大部分，表现为成长种群，CI最早出现在陆冰缘站的冰下推测生殖发生在十月至十一月间。巨锚哲水蚤的无节幼体集中出现于低纬度区，并没有发现有桡足幼体出现。

牡蛎染色体的分子生物学分析

本研究应用显带技术和荧光原位杂交(Fluorescence in situ hybridization，FISH)技术，鉴定了牡蛎的染色体；应用FISH方法定位了一系列的重复序列和大分子的P1克隆DNA；制备了染色体特异性探针。应用FISH特异性探针成功地鉴定了长牡蛎的三体10。结果如下：1．分析了G带和C带在美洲牡蛎染色体上的分布。G带在每一条染色体上的带型不同，某些染色体间(如第1对和第4对染色体，第7对和第9对染色体)的带型差别不是很明显。G带型容易受染色体收缩程度的影响。C带型重复性较好，染色体带型较清楚，分布在染色体的端粒区域和着丝粒区域。G带和C带带型能够用来鉴定牡蛎的染色体，但是重复性低和带型差异不显著，并不适合常规的染色体鉴定。2．早期胚胎和担轮幼虫制备的染色体适合于FISH分析。染色体制备方法重复性好，可适用于其它贝类的染色体制备。3．研究了重复序列基因--rDNA的定位：1)18S-5.8S rDNA在研究的五种巨蛎属Crassostrea牡蛎均只有一个位 点。太平洋种(C.gigas，C. ariakensis和C. plicatula)中，杂交信号位于最短的染色体一第10对染色体长臂的端粒区域，在大西洋种(C. virginica和C. rhizophorae)中，同一序列定位在第2对染色体短臂的端粒区域。2)18S-28S rDNA在两种蛤中有两个位点。rDNA探针定位在侏儒蛤(Mulinis Lateralis)的第15对和第19对染色体的端粒区域，同一序列定位在硬壳蛤(Mercenaria mercenaria)的第10对染色体的长臂和第12对染色体短臂的端粒区域。信号强度在两对染色体之间有差异。 3)5s rDNA位于美洲牡蛎的第5对染色体的短臂上靠近着丝粒区域和第6 对染色体的短臂的中间区域。信号强度在两对染色体之间没有显著差异。5S rDNA探针可以作为鉴定和识别第5对和第6对染色体的特异性探针。4．研究了一些重复序列的定位1)两个短的重复序列1G8，1P2均产生很强的荧光信号分布在美洲牡蛎所有的染色体上。在低严谨条件下，这些序列均产生很强的信号散布在所有的染色体上。在高严谨条件下，信号强度大大减弱，但是信号仍散布在所有的染色体上。这些重复序列散布在美洲牡蛎的整个基因组中。2)高度重复序列Cgl70产生的信号分布在长牡蛎的7对染色体的着丝粒区域，没有发现间区信号。在第1对，第2对，第4对和第7对染色体上的荧光信号强且稳定。在第5对，第8对和第10对染色体上的信号相对弱且不稳定。在剩余的染色体上(第3对，第6对和第9对染色体)没有检测到荧光信号。结果表明此卫星序列是一个着丝粒卫星序列。在美洲牡蛎的染色体上没有检测到荧光信号，表明了这个着丝粒卫星序列在这两种牡蛎中的分布存在着显著的差异。3)脊椎动物端粒序列(TTAGGG)n的FISH信号局限在四种双壳贝类(美洲牡蛎，the mangrove oyster，硬壳蛤，侏儒蛤)所有染色体的端粒区域，没有发现间区信号的存在。研究结果与已报道的研究结果表明脊椎动物端粒序列或许存在于所有双壳贝类的染色体末端。双壳贝类是目前研究过的唯一含有脊椎动物端粒序列DNA的无脊椎动物。4)研究了RAPD探针在美洲牡蛎染色体上的定位。大多数RAPD探针产生了多个信号散布在间期细胞核和所有的染色体上。引物OPX-03，OPX-04，OPX—06，OPG-02，OPM—04，OPM-11，0PS-02制备的探针在适宜的条件下产生特异性荧光 信号，分布在牡蛎的特定的染色体上。PCR特异性带产生的探针OPX—06—310和0PG-02—300产生了特异性的荧光信号：OPX—06—310产生的信号位于第5对染色体的短臂的近端粒区域，0PG—02—300探针定位到第3对染色体的短臂上。这两个探针是鉴定美洲牡蛎单条染色体的特异性探针。5．研究了大分子Pl克隆DNA(插入片断为80～100 kb)在美洲牡蛎染色体上的定位。Pl克隆DNA通过切口平移方法标记digoxigenin—11-dUTP用作FISH的探针。Cot-1 DNA作为竞争剂有效地抑制了Pl克隆序列中的重复序列产生的信号。杂交信号用fluorescein标记的anti—digoxigenin抗体来检测，用两层抗体rabbit-anti-sheep抗体和FITC anti—rabbit抗体来扩增信号。9个P1探针成功地定位在特定的染色体上。46—1探针杂交到第1对染色体的长臂靠近着丝粒区域；47-10探针定位到第2对染色体的长臂近端粒区域；Cvpl和48-13两探针定位到第3对染色体上：Cvpl位于短臂的端粒区域，48-13探针位于长臂的近着丝粒区域；48—10探针杂交到第4对染色体的长臂上；48-1探针杂交到第5对染色体长臂的近着丝粒区域；49-11探针位于第7对染色体长臂上；探针49-10和44-11位于第8对染色体长臂上。同时我们成功地将2个P1探针杂交到同一染色体分裂相中，进一步确定了Pl探针在美洲牡蛎染色体 上的定位。6．应用18S-28S rDNA探针成功地鉴定出长牡蛎非整倍体中的三体10。经鉴定AF-35，AF-39和AF-3三体家系属于三体10家系。rDNA探针分布在三条染色体上，即多出的一条染色体为染色体10。相应地在间期细胞核上有三个信号出现。AF-34和AF-36家系不属于三体10家系。rDNA探针分布在两条染色体上，相应地在间期细胞核上有两个信号出现。FISH和染色体特异性探针为非整倍体的鉴定提供了一个快速准确可靠的方法和途径。

改性粘土治理有害赤潮对生态环境的影响初探

本文以室内实验为基础，研究了改性粘土治理有害赤潮的方法对生态环境的影响。研究结果表明：有机和无机改性粘土对溶解氧（DO）、化学耗氧量（COD）、pH等主要水质因子均有改善作用；对营养盐，尤其是磷酸盐有一定的吸附作用，在外加磷酸盐为0-0.3μmol/ml范围内，吸附量随水体中磷酸盐浓度的增加而增大，有机改性粘土对海水中磷酸盐的吸附能力为：有机改性粘土Ⅰ>有机改性粘土Ⅱ>有机改性粘土Ⅲ。通过有机改性粘土对磷酸盐的吸附－再释放情况，进一步探讨了其磷酸盐的释放对赤潮异弯藻（Heterosigma akashiwo）、东海原甲藻（Prorocentrum donghaiense）等赤潮生物生长的影响。实验结果显示，经过有机改性的粘土有利于提高其对磷酸盐的吸附能力，降低对磷酸盐的解吸率，利用有机改性粘土治理赤潮可以缓解海水富营养化程度，即使被吸附的部分磷酸盐能缓慢释放，但仍不足以维持赤潮生物的正常生长。无机改性粘土对磷酸盐和硝酸盐与有机改性粘土有相似的吸附性能。其添加剂PAC的重金属含量符合沉积物排放要求，不会对环境造成压力。 其次，以太平洋牡蛎(Crassostrea gigas)幼贝为对象，研究了有机改性粘土Ⅱ和无机改性粘土在对海洋底栖生物的影响。急性毒性试验中二者对牡蛎幼贝的半致死浓度（LC50）分别为4.62 g/L和2.67 g/L，均远远大于改性粘土去除赤潮藻的浓度。在能够有效去除赤潮微藻的粘土浓度条件下，经慢性毒性试验发现改性粘土对牡蛎幼贝成活率、生长和摄食略有降低，但无明显影响，在牡蛎幼贝鳃组织和消化道组织的超微结构中未发现机械损伤。 最后，研究了改性粘土在对去除有毒赤潮藻过程中对底栖生物的影响。以能产生麻痹性贝毒（PSP）的塔玛亚历山大藻（A. tamarense，ATHK）为例，研究了PSP在水体中和粘土沉积层中的分布，发现改性粘土去除塔玛亚历山大藻能有效降低水体中藻细胞浓度及其所携带的PSP毒素。由于藻细胞破裂, 虽然沉积层中藻细胞体内部分PSP毒素溢出，但通过塔玛亚历山大藻细胞不同组分对牡蛎的影响发现，细胞内容物和细胞碎片对牡蛎没有明显毒副作用，由此推断改性粘土对藻细胞絮凝沉降后,能降低对生物的不良影响。此外，室内实验模拟了去除赤潮生物塔玛亚历山大藻（A. tamarense，ATHK）、东海原甲藻 (P. donghaiense )、赤潮异弯藻 (H. akashiwo)过程中，比较改性粘土和赤潮微藻对生物体的影响发现改性粘土在去除赤潮藻过程中能提高牡蛎的存活率，进一步证明改性粘土在赤潮生物防控中，是一种有效的应急措施。