Laboratory observation on spawning,fecundity and larval development of amphioxus (Branchiostoma belcheri Tsingtaunese)

Although amphioxus is widespread in temperate and tropical seas, its population is diminishing because of environmental pollution. To keep the population of this evolutionarily important animal from diminishing, study on its reproduction and development is necessary. The main findings in this study on the spawning and fecundity of the amphioxus reared in laboratory and its larval development are as follows. 1. Water temperature markedly affected the spawning. It spawned only when water temperature reached 21°C. 2. Spawning of the amphioxus in laboratory was markedly extended. Initially, the amphioxus spawned at about 7:00 PM, but spawning time was postponed as spawning days went on. 3. The number of eggs produced by a female ranged from 1400 to 12800, average of 5800. This also represents the fecundity of the amphioxus because it shedded all eggs within the ovary at a time. 4. During the first few months of life of the amphioxus, its growth rate changed seasonally. The growth rate in summer and fall was greater than that in winter. 5. The pelagic larva became a benthic adult after 50 days. 6. The amphioxus reared in laboratory from fertilized eggs could produce fertile eggs and sperms. These findings can be a foundation for measures to address the problem of diminishing amphioxus population. Contribution No. 2274 from the Institute of Oceanology, Chinese Academy of Sciences This work was supported by CNNSF, and Chinese Academy of Sciences.     

Production of fertile eggs and sperms in laboratory-reared amphioxusBranchiostoma belcheri tsingtaunese

Althoug hamphioxus has been studied embryologically for over 130 years, little is known concerning the gonadal development of the laboratory-reared animal. The present study in which the amphioxus were sucessfully maintained in the laboratory for more than one year showed that they could produce fertile eggs and sperms like their wild counterparts. Project (39470091) supported by NSFC and partially by Shandong Natural Science Foundation Grant 93D0140.     

Antioxidant enzyme activities in different genders and tissues of amphioxus Branchiostoma belcheri tsingtauense

Information regarding antioxidant enzymes in amphioxus remains lacking, and this study was carried out to examine the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in different genders and tissues of amphioxus Branchiostoma belcheri tsingtauense. Results show that (1) CuZn-SOD, CAT and GPX activities in the whole amphioxus B. belcheri tsing- tauense were basically at the same levels in male and female amphioxus, whereas both T-SOD and Mn-SOD activities in male amphioxus were significantly higher than that in the female (P<0.05); (2) The testis had significantly higher T-SOD and CuZn-SOD activities than the ovary (P<0.05); (3) CuZn-SOD activity was undetectable in the guts of male and female amphioxus; (4) For both male and female am- phioxus, the activities of CAT and GPX in the gonads including testis and ovary were the lowest (P<0.05) among the tissues examined; (5) The gut and gill had the same level GPX activities while the gut had a higher CAT activity; (6) There was no clear difference in CAT and GPX activities in the corresponding tissues between male and female amphioxus. The study on SOD, CAT and GPX activities in different genders and tissues of the protochordate provides data for future comparison of amphioxus antioxidant enzymes with those of invertebrates and vertebrates.     

MORPHOLOGICAL AND FUNCTIONAL STUDIES ON THE EPIDERMAL CELLS OF AMPHIOXUS (BRANCHIOSTOMA BELCHERI TSINGTAUENSE) AT DIFFERENT DEVELOPMENTAL STAGES

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Initiation of primary cell culture from amphioxus Branchiostoma belcheri tsingtauense

Amphioxus, a cephalochordate, is an important model fish for studies in evolution and comparative biology. A successful cell culture from amphioxus tissues in vitro would help understanding some basic issues. To determine the optimal culture conditions for proliferation of amphioxus cells, primary cultures were initiated from buccal cirri, tail, gill, gut and metapleural fold of amphioxus Branchiostoma belcheri tsingtauense. The media tested were L-15, F-12, M 199, MEM, DMEM, PRMI 1640 and LDF, each was supplemented with 20% fetal bovine serum. The optimal conditions include tail tissue cultured in L-15 or F-12 with supplement of 20% FBS and 1.5% NaCl at about 25°C.     

Morphological and functional studies on the epidermal cells of amphioxus (Branchiostoma belcheri tsingtauense) at different developmental stages

Epidermal cells of amphioxus at different developmental stages were investigated by electron microscopy and colloidal carbon tracing experiments. Amphioxus epidermal cells showed different ultrastructural characteristics at larval and adult stages. The epidermal cells at all larval stages studied (24–96 h) had numerous vesicles containing electron dense materials in their apical cytoplasm. In tracing experiments, carbon particles were found in apical vesicles and interoellular spaces. Under scanning electron microscope, many crater-like protrusions were observed on the surface of the cells. These results indicated that amphioxus larval epidermal cells may be capable of endocytosis. The epidermal cells of 3-month and adult amphioxus were obviously secretory ones characterized by well-developed peripheral filaments, a prominent Golgi apparatus and abundant apical secretory vesicles. This study also showed that adult amphioxus body surface mucus contained lectin that could agglutinate human red blood cells. The authors propose that the epidermal cells of amphioxus larva and adult may contribute to the immune defense of the amimal by different means. Project 3860811 supported by NSFC and study also supported by the Shandong Natural Science Foundation (Grant No. 92D1144).     

Initiation of primary cell culture from amphioxus <Emphasis Type="Italic">Branchiostoma belcheri tsingtauense</Emphasis>

Amphioxus, a cephalochordate, is an important model fish for studies in evolution and comparative biology. A successful cell culture from amphioxus tissues in vitro would help understanding some basic issues. To determine the optimal culture conditions for proliferation of amphioxus cells, primary cultures were initiated from buccal cirri, tail, gill, gut and metapleural fold of amphioxus Branchiostoma belcheri tsingtauense. The media tested were L-15, F-12, M 199, MEM, DMEM, PRMI 1640 and LDF, each was supplemented with 20% fetal bovine serum. The optimal conditions include tail tissue cultured in L-15 or F-12 with supplement of 20% FBS and 1.5% NaCl at about 25°C. Supported by Doctoral Initial Fund of Ludong University (No.43304)     

Artificial culture of amphioxus (Branchiostoma belcheri tsingtauense)

Mature amphioxus (Branchiostoma belcheri tsingtauense) collected by trawl in June, 1994 from Qingdao Shazikou sea area bottom sands, were cultured under controlled indoor conditions, where they spawned and their eggs were fertilized and hatched into larvae which gradually settled at the bottom and dug into the sands after 40-50 days culture in a water trough outdoors. The total survival rate of the larvae(LSR)was 5.5% before they went into the sands, 1.9% in 4 months, 0.7% in 10 months. The survival rate of the young fish which had dug into the sands (YSR) was 35.6% in 4 months, and 12.6% in 10 months. The amphioxus number tended to be constant from the 11th month on. In the first five months after incubation, the amphioxus body length increased by an average of about 1.5 mm a month, and about 0.4 mm a month from Dec. to May of next year. The maximum length after ten months was 24 mm; the average was about 11 mm. After two years culture in the water trough, the maximum length could reach 34-35 mm, when the amphioxus gonads began to develop.     

Interplay between amphioxus complement with sea bass macrophages: Opsonic activity of amphioxus humoral fluids

Previous studies have shown the existence of a complement system in the amphioxus Branchiostoma japonicum. However, whether it has an opsonic activity similar to that of vertebrates remains unknown. We demonstrated that the humoral fluid (HF) of amphioxus promoted the phagocytosis of yeast cells with sea bass (Lateolabrax japonicus) macrophages, whereas the C3-depleted and heated HF significantly lost the phagocytosis-promoting capacity. In addition, the precipitation of factor B (Bf) led to a marked loss of opsonic activity. Moreover, C3 fragments in the HF were found to bind to yeast cell surfaces. The results indicate that the amphioxus complement system is an important element involved in the opsonic activity, which promotes the sea bass macrophage phagocytosis by tagging yeast cells with C3 fragments via the activation of alternative complement pathway.     

INDUCTION OF ECTODERMAL CELLS FROM VEGETAL-ENDODERMAL BLASTOMERES OF AMPHIOXUS(BRANCHIOSTOMA BELCHERI TSINGTAUNESE) EMBRYOS BY THE CALCIUM IONOPHORE A23187

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Developmental expression pattern of calmodulin gene in amphioxus Branchiostoma belcheri tsingtauense

We investigated the developmental expression pattern of AmphiCaM in cephalochordate amphioxus (Branchiostoma belcheri tsingtauense). We cultured and sampled the animals at different developmental stages (eggs and larvae), and used in-situ hybridization and northern blotting to document the spatial and temporal changes in AmphiCaM expression. The alimentary tract dominates the development from the late neurula stage to the adult stage. AmphiCaM expression increased significantly in the alimentary tract during the late neurula stage and remained elevated in the adults. Our results indicate that AmphiCaM is involved in the differentiation of the alimentary tract in amphioxus; and furthermore, provide an insight into the change in function of CaM genes during evolution.     

Induction of ectodermal cells from vegetal-endodermal blastomeres of amphioxus (Branchiostoma belcheri tsingtaunese) embryos by the calcium ionophore A23187

Timing of vegetal-endodermal cell determination in amphioxus embryos remains uncertain. We tentatively tested effects of A23187, the calcium ionophore, on the development of vegetal blastomeres isolated at the 16-cell stage. It was found that when vegetal blastomeres committed to endoderm were treated with A23187 prior to gastrulation, they were transformed into ectodermal cells as evidenced by the cell morphology and function characteristic of epidermis. However, the developmental fate of the same blastomeres untreated or treated with DMSO at the same stage or of those treated with A23187 after gastrulation remained unchanged. Thus, vegetal-endodermal cells in amphioxus embryos are not irreversibly determined before the gastrula stage, and artificial increase in intracelluar Ca²⁺ concentration can induce transdetermination of the predetermined endodermal cells into ectodermal cells. Project 39470091 supported by NSFC and partly supported by Shandong Natural Science Foundation, Grant No.93D0140.     

Expression and localization of a novel phosducin-like protein from amphioxus Branchiostoma belcheri

A full length amphioxus cDNA, encoding a novel phosducin-like protein (Amphi-PhLP),was identified for the first time from the gut cDNA library of Branchiostoma belched. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histocbemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-NIIAmphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri.     

Expression and localization of a novel phosducin-like protein from amphioxus <Emphasis Type="Italic">Branchiostoma belcheri</Emphasis>

A full length amphioxus cDNA, encoding a novel phosducin-like protein (Amphi-PhLP), was identified for the first time from the gut cDNA library of Branchiostoma belcheri. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histochemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-N1/Amphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri. Supported by the Pilot Projects of Knowledge Innovation Project of Chinese Academy of Sciences (No. KZCX2-YW-211-03 and MGE2008KG06)     

湛江海域文昌鱼(Branchiostoma)线粒体Cyt b基因的全序列分析

测定了邻近湛江的茂名海区和硇洲岛海域文昌鱼各2个样本线粒体Cytb基因全序列,以尾索动物亚门的2种海鞘(Ciona spp.)和脊椎动物亚门的七鳃鳗(Petromyzon marinus)作为外群,利用分子系统学方法与GenBank收录的14个文昌鱼(Branchiostoma)的Cytb基因全序列进行了比较,UPGMA法构建了系统发生树。全序列分析表明:主产于大西洋(批针文昌鱼B.lanceolatum和佛罗里达文昌鱼B.floridae)、厦门文昌鱼(B.belcheri belcheri Gray)和湛江文昌鱼线粒体Cytb基因全序列长度为1143bp,终止密码子同为UAG;主产于青岛文昌鱼(B.belcheri tsingtaunese)和日本文昌鱼为1141bp,以T结尾,转录过程中构成UAA终止密码子。系统分析表明:16个文昌鱼样品体现出太平洋和大西洋、太平洋南北明显分化。确认采自茂名和硇洲岛的文昌鱼为厦门文昌鱼(B.belcheribelcheriGray);支持将白氏文昌鱼青岛亚种(B.belcheri tsingtaunese)提升为种,命名为日本文昌鱼(B.japonicus)。     

Gel microbead cultivation with a subenrichment procedure can yield better bacterial cultivability from a seawater sample than standard plating method

A gel microbead (GMD) cultivation method was employed to cultivate microorganisms from an amphioxus breeding zone in Qingdao, P. R. China. The culture results were compared with those by standard plating method. In the GMD-based method, the microcolony-forming GMDs were sorted by fluorescence-activated cell sorting (FACS). To further get pure cultures, a subsequent enrichment culture and a streaking purification procedure were conducted on marine R2A medium. Eighty bacterial strains isolated by the GMD-based method were randomly selected for sequencing. These isolates belonged to Alphaproteobacteria (33%), Gammaproteobacteria (44%), Bacteroidetes (11%), Actinobacteria (5%), Firmicutes (5%), Epsilonproteobacteria (1%), and Verrucomicrobia (1%), the last two groups being usually difficult to culture. The 16S rRNA gene sequences revealed a diverse community with 91.1%-100% of the bacterial rRNAs similarities. Thirteen strains were sharing 16S rRNA gene sequence which was less than 97% similar to any other rRNA genes currently deposited in TYP16S database. Seventy isolates derived from the standard plating method fell into 4 different taxonomic groups: Alphaproteobacteria (9%), Gammaproteobacteria (81%), Bacteroidetes (7%) and Firmicutes (3%) with a 16S rRNA gene sequence similarities between 95.8%-100%, in which only 3 strains were sharing 16S rRNA gene sequence of less than 97%. The results indicated that the GMD-based method with subenrichment culture yielded more taxonomic groups and more novel microbial strains, including members of previously rarely cultured groups, when compared with the standard plating method, and that this method markedly improved the bacterial cultivability.     

校园网络GIS教学科研平台的建设与管理探索

根据GIS教学实践的应用需求与GIS实验室建设的发展趋势,以清华大学校园网络GIS教学科研平台的建设与管理为例,从建设历程、管理体系、管理流程、应用成果等四个方面,探讨校园网络GIS教学科研平台的构建、管理与应用,旨在促进我国高校GIS实验室的建设,推动GIS教学实践的发展.     

没有围墙的GIS实验室

美国前副总统戈尔认为数字地球可以变成一个“没有围墙的实验室”,那么什么是“没有围墙的实验室”,如何建设 ?本文在对当前数字地球研究成果分析的基础上 ,指出“没有围墙的实验室”的核心就是“没有围墙的 GIS实验室”.     

基于DSP6000的实验室安全控制系统研究

以TMS320DM642为核心构建一个实验室安全控制系统,通过摄像机采集视频图像,将图像传输给DM642进行图像处理,包括获取背景图像、差影处理、图像分割、运动目标检测、安全控制,并将处理结果显示在液晶屏上.实验验证,系统能够实现实验室条件下的安全控制.     

不同甘蔗品种锤度与糖分的关系

使用大田实验数据和实验室数据建立了实验室锤度和田间锤度与甘蔗糖分关系的数学模型。统计分析表明 ,实验室锤度、田间锤度与糖分间均存在显著的线性关系 ,并且实验室锤度———糖分关系与大田锤度———糖分关系存在显著性 (α =0 .0 5 )差异 ;而不同甘蔗品种间的田间锤度———糖分关系无显著性差异 (α =0 .0 5 )。故不同的取样方法应使用不同的方程来估计甘蔗糖分 ;而不同甘蔗品种间田间锤度———糖分关系可用同一模型来描述。对三个供试甘蔗品种应用该模型进行实测验证 ,实测与预测值的相对误差均在 7%以内。