Cloning, characterization and expression of ferritin subunit from clam Meretrix meretrix in different larval stages

Shell formation is one of the important events during larval development and metamorphosis in bivalves. However, the molecular mechanisms and environmental cues regulating shell initiation and growth are unclear. Here, we report that ferritin, a principal protein for biological iron storage and metabolism, might play a role in larval shell development of the bivalve mollusk Meretrix meretrix. A full-length ferritin subunit cDNA, named as MmeFer, was cloned and characterized. The MmeFer mRNA expression in different developmental stages, from trochophore to post larvae, was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). MmeFer mRNA expression in larvae of later developmental stages increased at least 8-fold following trochophores. Moreover, the temporal and spatial expressions of MmeFer mRNA were examined by whole mount in situ hybridization. In the trochophore stage, MmeFer was detectable where it was supposed to be for shell initiation. In the later developmental stages, MmeFer was found near digestive glands and mantle that secret larval shell. MmeFer expression was also detected in larvae cultured in artificial seawater with different iron concentrations ranging from 0 to 100 mu M. These results suggest that ferritin may play a role in the shell formation of mollusks. (C) 2009 Elsevier Inc. All rights reserved.

Effect of stocking density on growth, settlement and survival of clam larvae, Meretrix meretrix

To determine the optimal larval density for hatchery culture of the clam Meretrix meretrix, experiments with stocking densities of 5, 10, 20, 40 and 60 larvae ml(-1) were designed, which included the developmental stages from D-veliger to 8 days postsettlement. Shell length, settlement time and survival rate of the larvae were recorded. Results showed that, at each sampling time, larvae reared at the highest density had the smallest mean size, whereas larvae reared at the lowest density had the largest mean size. Statistical differences in mean shell length at different stocking densities appeared from day 2, and greater differences occurred with increased culture time. Specific growth rate (SGR) in the rapid growing stage (day 0-3) was negatively correlated with density; however, no correlation was found between SGR and density in the slow growing stage (days 3-7). Settlement time was prolonged and shell length of settled larvae decreased as density increased. However, larval survival rate (74.8-79.1%) was independent of stocking density. Results showed that a high stocking density, in the designated range, is feasible for larval culture of the clam M. meretrix. However, for large-scale culture, in the interest of costs and safety, a stocking density of 10-20 larvae ml(-1) is recommended. (c) 2006 Elsevier B.V. All rights reserved.

Chromosomal mapping of major ribosomal rRNA genes in the hard clam (Mercenaria mercenaria) using fluorescence in situ hybridization

Karyotype and chromosomal location of the major ribosomal RNA genes were studied in the hard clam (Mercenaria mercenaria Linnaeus) using fluorescence in situ hybridization (FISH). Metaphase chromosomes were obtained from early embryos. Internal transcribed spacers (ITS) between major RNA genes were amplified and used as FISH probes. The probes were labeled with digoxigenin-11-dUTP by polymerase chain reaction and detected with fluorescein-labeled anti-digoxigenin antibodies. FISH with the ITS probes produced two to four signals per nucleus or metaphase. M. mercenaria had a haploid number of 19 chromosomes with a karyotype of seven metacentric, four metacentric or submetacentric, seven submetacentric, and one submetacentric or subtelocentric chromosomes (7M + 4M/SM + 7SM + 1SM/ST). Two ITS loci were observed: one located near the centromere on the long arm of Chromosome 10 and the other at the telomere of the short arm of Chromosome 12. FISH signals on Chromosome 10 are strong and consistent, while signals on Chromosome 12 are variable. This study provides the first karyotype and chromosomal assignment of the major RNA genes in M. mercenaria. Similar studies in a wide range of species are needed to understand the role of chromosomal changes in bivalve evolution.

Effects of diet, stocking density, and environmental factors on growth, survival, and metamorphosis of Manila clam Ruditapes philippinarum larvae

A series of experiments was conducted to evaluate the effects of diet, stocking density, and environmental factors on growth, survival, and metamorphosis of Manila clam Ruditapes philippinarum larvae. These experiments examined the following factors: diet (Isochrysts spp., Chlorella spp., and a mixture of Isochrysis spp. and Chlorella spp. [ 1: 1 w/w]), stocking density (5, 10, 15, and 20 larvae ml(-1)), light intensity (un-shaded, partially shaded, and fully shaded), water filtration (unfiltered and sand-filtered), water exchange (50% and 100% once every other day, 25%, 50%, and 100% once daily; 50% and 100% twice daily), and the use of substrate (with and without sand as the substrate). Results indicated that Chlorella spp. could replace 50% of Isochrysis spp. as a food source for the Manila clam larvae without affecting growth, survival, and metamorphosis. Larval growth decreased significantly with increasing stocking density. A density of 5-10 larvae ml(-1) appeared to be optimal for normal growth of Manila clam larvae. Neither diet nor stocking density used in the study had a significant effect on larval survival. Under partially shaded (light intensity = 1000-5000 lx) and fully shaded (light intensity <500 lx) conditions, larval growth was significantly faster than under direct sunlight (un-shaded). A water exchange rate of 50% twice daily provided optimum larval growth. Larvae grew significantly faster in the unfiltered water than in the sand-filtered water. Using sand as the substrate in the culture system significantly depressed the metamorphosis rate. The type and particle size of sand used as the substrate did not significantly affect growth and metamorphosis rates of the larvae. (C) 2005 Published by Elsevier B.V.

A new three-phase culture method for Manila clam, Ruditapes philippinarum, farming in northern China

Studies on reproduction, hatchery management, and culture of Manila clams Ruditapes philippinarum were carried out in an attempt to optimize their culture conditions and techniques. Results from these studies led to the development of a three-phase culture method for Manila clam farming in northern China. The key components of the new method were: 1) early spawning and over-wintering indoors (greenhouse); 2) optimized larval culture conditions and techniques; 3) juvenile rearing in shallow, fertilized nursery ponds; 4) optimized stocking size and density and substrate for mudflat grow out. Broodstock were maturated indoors for a month from early April to early May. Primarily because of higher water temperatures in the greenhouse the clams spawned more than one month earlier than in the natural environment. From May to July, juveniles were reared for 1-2 months indoors to a size of 2.0-3.0 mm in shell length before being moved to outdoor, pre-disinfected, nursery ponds. Juveniles were then reared in the nursery ponds for one month to about 1.0 cm before being transferred to the mudflat for grow out. Juvenile clams in nursery ponds grew considerably faster than in the natural environment probably because of higher temperatures and more abundant natural food. During grow out, the clams were reared for 4-7 months until they reached a market size (3.0-3.3 cm). Juveniles produced after August were over-wintered in the greenhouse in which the water temperature was about 3 degrees C higher than that of the outdoor environment. Juveniles grew at an average rate of > 20 mu m day(-1), while in the natural environment no growth was observed during winter because of low temperatures. Juveniles in the greenhouse grew to 2-3 mm by the following March before being moved into outdoor nursery ponds. The three-phase culture method not only shortened the production period from spawn to market size from 24-36 months to about 10-14 months, but also prolonged the spawning season from 2 to 7 months, resulting in increased production of seed and market-size clams. Compared with the traditional method, the new method could increase the yield of market-size clams by 10-11 times, and increase the profit per ha mudflat by as much as 124 times and the profit per kg market-size clams produced by 13 times. (c) 2006 Elsevier B.V. All rights reserved.

Indole and carbazole alkaloids from Glycosmis montana with weak anti-HIV and cytotoxic activities

A diprenylated indole, (E)-3-(3-hydroxymethyl-2-butenyl)-7-(3-methyl-2-butenyl)-1H-indole (1), and six known carbazole alkaloids were isolated from the twigs and leaves of Glycosmis montana Pierre (Rutaceae). Their structures were determined on the basis of analysis of spectral evidence including 1D and 2D NMR and MS. The alkaloids (1-3) exhibited weak to moderate take in vitro inhibitory activity against HIV replication in C8166 cells, and they (as well as carbalexine A and B) had cytotoxic activity against the human leukaemia cell line CCRF-CEM. (c) 2005 Elsevier Ltd. All rights reserved.

Life history and production of Sphaerium lacustre (Mollusca : Sphaeriidae) in a shallow subtropical Chinese lake

A fingernail clam, Sphaerium lacustre, was studied in subtropical Lake Donghu from June 1999 to May 2000. The S. lacustre population was characterized by a single annual reproduction period starting in March and ending in October; the population comprised three size groups, of which the 1999 cohort was dominant. The annual average density and biomass were 100.2 ind./m(2) and 12.11 g/m(2), respectively The annual production was 43.02 wet weight g/m(2), and the corresponding annual production/biomass ratio was 3.55.

Knocking-Down Cyclin A(2) by siRNA Suppresses Apoptosis and Switches Differentiation Pathways in K562 Cells upon Administration with Doxorubicin

Cyclin A(2) is critical for the initiation of DNA replication, transcription and cell cycle regulation. Cumulative evidences indicate that the deregulation of cyclin A(2) is tightly linked to the chromosomal instability, neoplastic transformation and tumor proliferation. Here we report that treatment of chronic myelogenous leukaemia K562 cells with doxorubicin results in an accumulation of cyclin A(2) and follows by induction of apoptotic cell death. To investigate the potential preclinical relevance, K562 cells were transiently transfected with the siRNA targeting cyclin A(2) by functionalized single wall carbon nanotubes. Knocking down the expression of cyclin A(2) in K562 cells suppressed doxorubicin-induced growth arrest and cell apoptosis. Upon administration with doxorubicin, K562 cells with reduced cyclin A(2) showed a significant decrease in erythroid differentiation, and a small fraction of cells were differentiated along megakaryocytic and monocyte-macrophage pathways. The results demonstrate the pro-apoptotic role of cyclin A(2) and suggest that cyclin A(2) is a key regulator of cell differentiation.

Molecular cloning and functional analysis of cathepsin B in nutrient metabolism during larval development in Meretrix meretrix

Seed rearing is an important part in large scale clam culture industry. Since the nutritional history affects early development in bivalve, the condition of larval nutrition plays a key role in successful seed rearing. So far, the molecular mechanism of nutrient uptake in bivalve larvae is unclear. As one of the important proteolytic enzymes, cathepsin B of several organisms has been reported to be involved in digestion. We intended to analyze whether cathepsin B is involved in larval nutrient metabolism in the economic bivalve, clam Meretrix meretrix. The full length of M. meretrix cathepsin B (MmeCB) cDNA was cloned, which is 1647 bp with an open reading frame of 1014 bp. The deduced amino acid sequence encoded a preproenzyme of 337 residues with Cys-114, His-282 and Asn-302 composing cathepsin B activity center. The temporal and spatial expressions of MmeCB mRNA were examined from trochophore to post larva stages by whole mount in situ hybridization. In trochophore stage, no detectable signal was found. In the later three stages, MmeCB mRNA was detected in the digestive gland, suggesting a possible role of MmeCB in digestion. Moreover, MmeCB mRNA was also observed in the epidermal cells in D-veligers. Cathepsin B specific inhibitor (CA074 methyl ester) was applied to block the activity of cathepsin B in unfed larvae. The average shell lengths of treated larvae were smaller than that in control groups. The results of mRNA epidermal distribution and inhibitor treatment in D-veligers indicated that MmeCB may be also associated with other pathway of nutrient metabolism in larval epidermis. The overall results in this paper revealed that MmeCB might play a role in larval nutrient metabolism. (C) 2008 Elsevier B.V. All rights reserved.

硬壳蛤性腺发育和卵母细胞成熟机理初步研究

采用体外药物诱导的方法，研究了5-羟色胺（5-hydroxytryptamine，5-HT）诱导的硬壳蛤卵母细胞成熟过程中cAMP信号通路的作用。结果表明，5-HT （0.01—100µM）均能够显著地诱导硬壳蛤卵母细胞的成熟。磷酸二酯酶抑制剂—咖啡因、茶碱和IBMX（3-异丁基-1-甲基黄嘌呤）可以单独抑制卵母细胞的自发成熟，但效果不显著。10mM的咖啡因和茶碱以及5mM的IBMX能够显著地抑制5-HT的诱导效果。dbcAMP（双丁酰基环腺苷一磷酸）不但能够抑制卵母细胞的自发成熟，而且还可以抑制5-HT诱导的成熟。因此，cAMP信号通路参与了5-HT诱导的硬壳蛤卵母细胞的成熟过程，并且该信号通路起着负调控的作用。 研究了PLC（磷脂酶C）和PKC（蛋白激酶C）的激活剂/抑制剂对5-羟色胺诱导的卵母细胞成熟的影响。高浓度的新霉素（PLC抑制剂）可以抑制5-HT诱导的卵母细胞的成熟，而DMBA（9,10-Dimethy-1,2-benzanthracene，9,10–二甲基胆蒽，PLC激活剂）则能够促进成熟。PMA（phorbol 12-myristate 13-acetate，佛波十四烷酸乙酸酯，PKC激活剂）能够抑制5-HT诱导的成熟，而Spingosine（PKC抑制剂）则可以促进卵母细胞的成熟。从而推测，5-HT诱导的卵母细胞成熟需要磷脂酰肌醇信号通路的激活。PLC浓度的降低能够抑制5-HT诱导的卵母细胞成熟；PKC浓度的降低则会促进卵母细胞的成熟。因此，在硬壳蛤卵母细胞的成熟过程中，PLC起促进的作用，DAG（二酰肌甘油）–PKC通路则起抑制的作用。 细胞外高浓度Ca2+能够促进硬壳蛤卵母细胞的成熟，Ca2+离子载体A23187也可以促进硬壳蛤卵母细胞的成熟。1-100µM异搏定（Verapamil，钙离子通道阻断剂）能够抑制卵母细胞的成熟，而100µM的Verapamil能够完全抑制其成熟。上述结果表明细胞外Ca2+对硬壳蛤卵母细胞的成熟是必需的，而且起到促进卵母细胞成熟的作用。三氟拉嗪（TFP，Ca2+与CaM结合的拮抗剂）能够抑制卵母细胞的成熟，高浓度的三氟拉嗪（1mM）能够完全抑制卵母细胞的成熟。说明CaM起到促进卵母细胞成熟的作用。可见，Ca2+通过与CaM的相互作用，共同起到促进硬壳蛤卵母细胞成熟的作用。 5-HT诱导成熟的卵母细胞可以完成受精过程，其受精过程以及幼虫发育情况与正常受精发育过程类似，没有显著差异。高浓度的新霉素可以抑制受精过程，而茶碱和咖啡因对受精没有影响。从而推测，磷脂酰肌醇信号通路参与了硬壳蛤卵母细胞的受精过程，而cAMP信号通路可能没有参与受精过程。 发现硬壳蛤的性腺发育与我国常见的双壳类如泥蚶相似。硬壳蛤卵母细胞中卵黄粒主要由线粒体、高尔基液泡、内质网和微吞饮泡形成。

文蛤（Meretrix meretrix）幼虫生长发育相关基因的克隆和功能分析

贝类养殖是我国海水养殖业中最重要的组成部分之一。贝类人工大规模养殖中的关键环节是种苗的人工繁育，早期幼虫能否正常生长发育，对幼虫变态和变态后生长率及成活率有很大的影响，直接关系到生产产量和经济效益，所以，了解幼虫发育机制对于生产实践具有重要的指导意义。 文蛤（Meretrix meretrix）是一种在东亚各国沿海和滩涂地区广泛分布的双壳贝类，是我国一种重要的经济品种。本论文以文蛤幼虫为研究对象，分别对文蛤幼虫发育过程中贝壳形成相关的铁蛋白（MmeFer）、营养及变态相关的组织蛋白酶B（MmeCB）及变态过程中细胞凋亡相关的caspase三个基因进行了克隆，分析了基因及编码蛋白在担轮幼虫期（L1）、D形幼虫期（L2）、壳顶幼虫期（L3）和稚贝期（L4）的时空表达特征，解析了其可能的功能，并研究了相应酶类的特异性抑制剂作用对幼虫发育过程的影响，进行了目标蛋白的功能验证，详述如下： 研究结果显示，在文蛤胚胎发育到原肠胚时放入不含铁离子的人工海水中培养，发育成无壳的畸形，随着人工海水中铁离子添加浓度的升高，幼虫长出壳状组织接近正常状态；而发育到L1期幼虫放入不含铁离子的人工海水中培养却可以发育出正常的壳，推测铁和铁代谢相关蛋白在幼虫贝壳初始形成有重要的作用。根据构建的文蛤幼虫cDNA文库中提供的序列信息，从文蛤中克隆了与铁离子代谢密切相关的铁蛋白（MmeFer）的全长cDNA 序列；通过Real time PCR发现，MmeFer mRNA的表达量在贝壳形成前后有明显改变；整体原位杂交结果显示MmeFer mRNA在L1期的表达部位刚好是贝壳生成的起始部位，推断文蛤铁蛋白与文蛤幼虫贝壳初始形成密切相关。 利用文蛤幼虫cDNA文库中的EST信息在幼虫中克隆到文蛤组织蛋白酶B（MmeCB）全长cDNA序列；通过整体原位杂交分析发现MmeCB mRNA在L2至L4期幼虫的消化腺部位表达，而且在L2期的幼虫表皮也有表达，说明MmeCB可能和幼虫消化相关，而且可能参与幼虫从表皮摄取营养的过程。利用MmeCB特异性抑制剂（CA074Me）处理饥饿幼虫，发现其生长受到明显抑制，验证了MmeCB参与幼虫表皮营养代谢的推论。利用免疫组织化学技术，研究了MmeCB蛋白在文蛤幼虫中的时空分布，发现其在L2期幼虫表面和胃部有阳性信号，而在L3期，MmeCB在幼虫面盘基部有强烈的表达，提示MmeCB在文蛤幼虫中不仅起到营养的作用，而且可能和幼虫附着变态有关。利用CA074Me分别处理文蛤胚胎和变态期幼虫，发现抑制MmeCB的活性对胚胎发育和幼虫变态都有显著影响。研究结果提示MmeCB在幼虫发育各阶段均具有重要的生物学功能。 根据caspase在不同物种中的保守区域设计简并引物，在文蛤幼虫中扩增到cDNA序列片段；检测有活性的caspase在文蛤幼虫各发育时期的分布部位，发现其在L1至L3期幼虫中都有分布，说明整个幼虫形态变化过程中都有caspase的参与；细胞凋亡检测结果显示，幼虫主要发生细胞凋亡的部位在L3期幼虫的面盘，即变态过程中要退化的器官，说明细胞凋亡可能是文蛤幼虫变态过程中面盘退化的主要机制；用caspase特异性抑制剂处理变态前幼虫，发现幼虫变态率下降，初步验证了caspase在文蛤幼虫变态过程中的作用。 通过对上述三种基因的研究，分别探讨了文蛤幼虫发育阶段中的几个主要事件（L1期到L2期的贝壳形成、L2到L3期的幼虫营养摄食及L3到L4期的附着变态）相关的基因及其功能，为研究贝类生长发育调控的分子机理提供了新的线索。

Accumulation of petroleum hydrocarbons and heavy metals in clams (Ruditapes philippinarum) in Jiaozhou Bay, China

Accumulation and distributions of aliphatic and polyaromatic hydrocarbons (PAHs) and heavy metals were measured in tissues of the clam Ruditapes philippinarum collected from 5 sites in Jiaozhou Bay, Qingdao, China. The concentrations of total aliphatic hydrocarbon and PAHs ranged from 570 to 2 574 ng/gdw (gram dry weight) and from 276 to 939 ng/gdw, in the most and least polluted sites, respectively. The bio-accumulation of hydrocarbons and PAHs in the clams appeared to be selective. Aliphatic hydrocarbons were predominantly represented by short chain (< nC(23)) n-alkanes, suggesting that petroleum hydrocarbons were likely the major contamination source. The selective uptake of 3 and 4 ring PAHs, such as naphthalene, fluorene, phenanthrene, fluoranthene and pyrene, by the clams was probably related to the physiological and bio-kinetic processes that were energetically favorable for uptake of compounds with fewer rings. Accumulation of the metals Cd, Cu, Zn, Pb, Cr, Hg, and As in the clam tissues also showed high variability, ranging from 0.043 to 87 A mu g/gdw. Among the 7 detected metals, Zn, Cd, Cu, and As had a particularly high potential of accumulation in R. philippinarum. In general, a positive correlation was found between the tissue concentrations and sediment concentrations of hydrocarbons and of some metals. Our study suggests that moderate contamination with polyaromatic hydrocarbons, and low to moderate contamination with metals, currently exists for clam R. philippinarum in Jiaozhou Bay, in comparison with other regional studies. A long-term monitoring program is certainly needed for assessment of the potential ecological influence and toxicity of these contaminants of R. philippinarum in Jiaozhou Bay.

INVOLVEMENT OF CA(2+) SIGNALING PATHWAY DURING OOCYTE MATURATION OF THE NORTHERN QUAHOG MERCENARIA MERCENARIA

In many molluses, it has been found that Ca2+ signaling pathway is involved in the resumption of meiotic maturation in oocytes. To better understand the possible role of Ca2+ signaling pathway in regulating meiotic maturation in oocytes of the northern quahog Mercenaria mercenaria, free extracellular Ca2+, A23187 (calcium ionophore), verapamil (calcium channel blocker), and trifluoperazin (calmodulin antagonist) were used to incubate oocytes or serotonin-induced oocytes by pharmacological methods. Results show that extracellular Ca2+ (50 similar to 200 mM) and A23187 (1 similar to 10 mu M) can stimulate the meiotic maturation. In addition, verapamil (1 similar to 100 mu M) and trifluoperazin (10 similar to 1,000 mu M) could inhibit serotonin-induced oocyte maturation. Therefore, Ca2+ is essential for the reinitiation of meiotic maturation in oocytes of the northern quahog. Moreover, an increase i [Ca2+]i can promote meiotic maturation.

Investigation of heavy metals in sediments and Manila clams Ruditapes philippinarum from Jiaozhou Bay, China

Manila clams (Ruditapes philippinarum) and sediments were collected bimonthly during 2007 at five locations in Jiaozhou Bay near Qingdao, China, to determine heavy metal concentrations and to assess the validation of R. philippinarum as a metal biomonitor. Concentrations of heavy metals in clam soft tissues ranged between 0.75 and 3.31, 0.89 and 15.20, 5.70 and 26.03, 52.12 and 110.33, 10.30 and 72.34, 9.64 and 28.60, and 3.15 and 52.75 mu g g (-aEuro parts per thousand 1) dry weight for Cd, Pb, Cu, Zn, Mn, Cr, and Ni, respectively. Most of the highest values occurred at the northeast bay and the lowest values occurred at the western part. Regarding seasonal variation, relatively high tissue metal concentrations were observed during October or December. A similar pattern was also found in habitat sediments. There was a strong correlation between the concentrations of Cd, Pb, Zn, Mn, Cr, and Ni in soft tissues and surrounding sediments. It is indicated that R. philippinarum could be used as a biomonitor for heavy metal contamination in Jiaozhou Bay.