栉孔扇贝大规模死亡病原学研究

用平板画线法从患病栉孔扇贝（Chlamys farreri）体内分离到了一种原核生物（简称QDP）。QDP可以在改进的液体培养基MEM（含2.2%NaCl，5%小牛血清）和脑心浸液（含2.2% NaCl）中生长；菌落在显微镜下（150×）为无色、透明的小点状；革兰氏染色阴性；菌体为圆形或近似圆形。QDP在发育过程中有两种状态，一种为未成熟阶段，直径小于100nm；另一种为成熟阶段，直径变化很大，最小约60nm，最大可达4µm以上。较小的个体有拟核、核糖体和新月状的空泡，未见细胞壁；较大的个体有细胞壁，胞内大部分被空泡充满，未见拟核和核糖体。栉孔扇贝组织超簿切片电镜观查证实QDP的存在。QDP的密度随着生长发育时间的不同而有所变化，繁殖高峰期密度较大。 建立了密度梯度离心结合滤膜过滤分离技术，优化人工培养条件。最适生长温度为23℃，最适生长pH值为7.4，最适生长盐度相当于细胞培养液所需的盐浓度（0.85%NaCl）。 提取的QDP核酸能被RNase A 降解，且没有检测到DNA。以PCR、RT-PCR扩增其16SrRNA基因序列片段，PCR反应没有扩增出扩增子，而RT-PCR则扩增出了16S rRNA基因序列片段，经测定其序列全长度为1430bp，经与GENEBANK中的16S rRNA片段比较分析，与6种不同科的微生物的同源率最高的为95%-95.47%。 采用温度梯度和病原浓度梯度回归感染实验方法，较为系统地研究了QDP的致病性。研究结果表明：QDP对栉孔扇贝有强烈的致病作用，高温（23℃以上）是其致病的必要条件，证实DQP是栉孔扇贝大规模死亡的病原体之一。

青蟹属的系统进化和中国沿海拟穴青蟹的群体遗传结构研究

为了进一步研究青蟹属系统进化的科学问题，并揭示我国东南沿海青蟹群体遗传结构和群体进化细节信息，本论文主要开展了以下两个方面的研究：（1）基于线粒体12S rRNA、16S rRNA和COI三种基因序列探讨中国东南沿海青蟹的种类归属与青蟹属的系统进化；（2）利用线粒体COI基因标记分析中国东南沿海拟穴青蟹的群体遗传结构。序列特征、遗传距离和系统进化分析结果都表明本文研究的青蟹均为S. paramamosain。NJ、BAYES和ML系统进化树显示S. paramamosain与S. tranquebarica互为姐妹种，S. olivecea应该是4种青蟹中最早分化出来的种类。10个地理群体130只拟穴青蟹的线粒体DNA（mitochondrial DNA，mtDNA）细胞色素氧化酶亚基I（COI）基因序列Mantel检验结果显示群体间的遗传分化程度与地理距离没有显著的相关性。分子进化中性检验结果表明自然选择在分子进化过程中起了重要作用，并暗示该物种在最近经历了一个快速的群体爆发及扩张事件。

鄂霍次克海天然气水合物区柱状沉积物LV39-18H微生物多样性研究

冷泉是指温度接近于海水，而以高于周围水环境浓度的烃类化合物（主要为甲烷）、硫化物或二氧化碳为主要成分，受地质构造或压力梯度作用渗出沉积物表层的流体。对冷泉沉积物中微生物群落的调查，有助于认识该极端环境中某些生理未知微生物类群的功能并理解微生物活动对整个系统的影响。 本文对从鄂霍次克海冷泉区采集获得的沉积物样品按深度划分得到的11个断层中的6个断层进行了总基因组的提取，利用16S rDNA作为分子标记，构建克隆文库并结合总有机碳、总氮、硫等环境因子对该样品中的细菌和古菌群落结构沿沉积物断层的分布情况进行研究，结果显示该沉积物中的细菌和古菌均具有高度多样性且显示出明显的成层分布： 1.细菌群落主要来自10个细菌门，优势门类为绿弯菌、未定门JS1、γ-、δ-变形菌，同时还发现浮霉菌、未定门OP8、放线菌、酸杆菌、拟杆菌、疣微菌的存在。我们还在分布于表层沉积物δ-变形菌类群中发现了占该层群落15%以上的SRB（硫酸盐还原菌）类群，这强烈提示着该沉积物环境中存在着AOM（甲烷厌氧氧化）过程。 2.古菌类群主要划分为DSAG、MBG-D、MCG、MGI、MBG-A和MHVG等类群。其中MBG-D类群沿断层的垂直分布与沉积物中硫含量表现出相似的变化趋势，这提示MBG-D类群可能参与该环境中硫相关的地质化学过程。

东、西太平洋深海沉积物细菌多样性研究

深海生物圈有着不同于陆地和浅海的典型特点，例如高压、低温、永久黑暗及寡营养，并且深海微生物具有特殊的代谢途径及庞大的生物量，这使得深海成为一个巨大的有待开发利用的生物资源宝库。 本文研究的样品分别取自东太平洋E272站位(12°36’39"N, 104°19’28"W)和西太平洋Ph05-5站位(16°04’93"N, 124º34’48"E)。E272站位距离东太平洋13°N海隆45km，水深3 191m；而Ph05-5站位地处西菲律宾海盆，在黑潮源区附近，位于西太平洋暖池区边缘，水深3 382m，并且Ph05-5岩芯一共包含了五个明显的火山灰层。 本文采用了末端限制性片段长度多态性分析（T-RFLP）和16S rRNA 基因文库分析的方法在小尺度上对东太平洋E272站位的沉积物样品进行细菌群落结构的研究。研究结果表明沉积物细菌群落结构在小尺度上存在明显的垂直变化。系统进化分析表明，该沉积物样品的细菌多样性较高，共包含9个主要的门类，包括变形菌门、绿弯菌门（绿色非硫细菌）、浮霉菌门、酸杆菌门、放线菌门（高G+C革兰氏阳性菌）、拟杆菌门、硝化螺旋菌门、以及两个待定的门类OP8和TM6。其中变形菌门细菌是一类在海洋中非常常见的细菌，广泛分布于各个海洋环境，在我们的文库当中发现了变形菌门的三个纲，包括α-、-、-变形菌纲。本项研究充分表明该沉积物环境中具有较高的细菌多样性，在小尺度上细菌群落垂直分布明显，其结果也可从侧面反映深海沉积物近表层处的环境条件在小尺度上的垂直变化显著。 对西太平洋暖池区沉积物样品的细菌群落的研究也采用16S rRNA 基因文库分析的方法。系统进化分析表明该沉积物样品细菌的多样性相对较低，一共包含了六个不同的门类，包括变形菌门、浮霉菌门、放线菌门、厚壁菌门（低G+C革兰氏阳性菌）、绿弯菌门、酸杆菌门。在这个沉积物样品中也发现了变形菌门的三个纲包括α-、-和-变形菌纲。聚类分析和系统进化分析都表明表层的细菌群落同其它8层的细菌群落存在明显的差异，并且其它8层包括5个火山灰层和3个远洋粘土层的细菌群落结构差异不大，推测火山灰成分不仅对火山灰层的细菌群落产生影响，而且可能通过扩散对整个沉积物的微生物群落结构都产生影响。表层可能由于沉积时间较晚所以受影响相对较小或表层本身不同于较深层次的理化条件而使表层群落存在较大差异。 对东、西太平洋不同环境下的两个深海沉积物样品的细菌多样性进行比较，结合其它研究发现变形菌门细菌在不同深海环境中都普遍存在，是深海不同环境的广适类群。另外，两个环境中的细菌多样性存在很大差异，东太平洋沉积环境中的细菌多样性要远高于西太平洋沉积环境中的细菌多样性，推测其最可能的原因是西太平洋沉积物火山灰成分对细菌群落的影响，致使其细菌群落与东太平洋远洋粘土沉积物细菌群落产生很大差异；另外，不同洋区的环境差异也应该是造成细菌群落差异的一个重要方面。

A bioaugmentation failure caused by phage infection and weak biofilm formation ability

Two biological aerated filters (BAF) were setup for ammonia removal treatment of the circulation water in a marine aquaculture. One of the BAFs was bioaugmented with a heterotrophic nitrifying bacterium, Lutimonas sp. H10, where the ammonia removal was not improved and the massive inoculation was even followed by a nitrification breakdown from day 9 to 18. The nitrification was remained stable in control BAF operated under the same conditions. Fluorescent in situ hybridization (FISH) with rRNA-targeted probes and cultivable method revealed that Lutimonas sp. H10 almost disappeared from the bioaugomented BAF within 3 d, and this was mainly due to the infection of a specific phage as revealed by flask experiment, plaque assay and transmission electron observation. Analyses of 16S rRNA gene libraries showed that bacterial groups from two reactors evolved differently and an overgrowth of protozoa was observed in the bioaugmented BAR Therefore, phage infection and poor biofilm forming ability of the inoculated strain are the main reasons for bioaugmentation failure. In addition, gazing by protozoa of the bacteria might be the reason for the nitrification breakdown in bioaugmented BAF during day 9-18.

Isolation and identification of bacteria associated with the surfaces of several algal species

We conducted this study to assess the diversity of bacteria associated with the surfaces of algae based on 16S rDNA sequence analyses. Twelve strains of bacteria were obtained from the surfaces of the following four species of algae: Gracilaria textorii, Ulva pertusa, Laminaria japonica, and Polysiphonia urceolata. The isolated strains of bacteria can be divided into two groups: Halomonas and Vibrio, in physiology, biochemical characteristics and 16S rDNA sequence analyses. The phylogenetic tree constructed based on 16S rDNA sequences of the isolates shows four obvious clusters, Halomonas venusta, Vibrio tasmaniensis, Vibrio lentus, and Vibrio splendidus. Isolates from the surface of P. urceolata are more abundant and diverse, of which strains P9 and P28 have a 16S rDNA sequence very similar (97.5%-99.8%) to that of V. splendidus. On the contrary, the isolates from the surfaces of G textorii, U. pertusa and L. japonica are quite simple and distribute on different branches of the phylogenetic tree. In overall, the results of this study indicate that the genetic relationships among the isolates are quite close and display a certain level of host species specificity, and alga-associated bacteria species are algal species specific.

Antimicrobial screening and active compound isolation from marine bacterium NJ6-3-1 associated with the sponge Hymeniacidon perleve

Twenty-nine marine bacterial strains were isolated from the sponge Hymeniacidon perleve at Nanji island, and antimicrobial screening showed that eight strains inhibited the growth of terrestrial microorganisms. The strain NJ6-3-1 with wide antimicrobial spectrum was identified as Pseudoalteromonas piscicida based on its 16S rRNA sequence analysis. The major antimicrobial metabolite, isolated through bioassay-guide fractionation of TLC bioautography overlay assay, was identified as norharman (a beta-carboline alkaloid) by EI-MS and NMR.

Isolation and characterization of novel marine Roseobacter clade members producing unique intracellular chromium-rich aggregates

The marine Roseobacter clade comprises one of the largest fractions of heterotrophic marine bacteria and accounts for about 16% of 16S rRNA gene clones retrieved from marine bacterioplankton. Their global distribution seems to be related to oceanic water masses and their environmental and biogeochemical properties. In this study, we report isolation and characterization of novel Roseobacter clade members from the Yellow Sea, China. Phylogenetic analysis of 16S rRNA gene sequences reveals that the new isolates (YSCB1, YSCB2, YSCB3 and YSCB4) are closely related to uncultured Arctic seawater bacterium R7967 (99.57-100% sequence identity) and to the cultured Roseobacter sp. DSS-1 (99.27-99.76% sequence identity) isolated from the southeastern coastal water of the USA. Interestingly, YSCB strains possess unique intracellular chromium-containing aggregates. Therefore, these novel Roseobacter clade members exhibit a peculiar property in mineral biogeneration. (c) 2006 Elsevier SAS. All rights reserved.

Isolation and characterization of a marine magnetotactic spirillum axenic culture QH-2 from an intertidal zone of the China Sea

Magnetotactic bacteria (MTB) are ubiquitous in aquatic habitats. Because of their fastidious requirements for growth conditions, only very few axenic MTB cultures have been obtained worldwide. In this study, we report a novel marine magnetotactic spirillum axenic culture, designated as QH-2, isolated from the China Sea. It was able to grow in semi-solid or liquid chemically defined medium. The cells were amphitrichously flagellated and contained one single magnetosome chain with an average number of 16 magnetosomes per cell. Phosphate and lipid granules were also observed in the cells. Both rock magnetism and energy-dispersive X-ray spectroscopy characterizations indicated that the magnetosomes in QH-2 were single-domain magnetites (Fe3O4). QH-2 cells swam mostly in a straight line at a velocity of 20-50 mu m/s and occasionally changed to a helical motion. Unlike other magnetotactic spirilla. QH-2 cells responded to light illumination. As a consequence of illumination, the cells changed the direction in which they swam from parallel to the magnetic field to antiparallel. This response appears to be similar to the effect of an increase in [O-2]. Analysis of the QH-2 16S rRNA sequence showed that it had greater than 11% sequence divergence from freshwater magnetotactic spirilla. Thus, the marine QH-2 strain seems to be both phylogenetically and magnetotactically distinct from the freshwater Magnetospirillum spp. studied previously. (C) 2010 Elsevier Masson SAS. All rights reserved.

Classification of jinjiang oysters Crassostrea rivularis (Gould, 1861) from China, based on morphology and phylogenetic analysis

The jinjiang oyster Crassostrea rivularis [Gould, 1861. Descriptions of Shells collected in the North Pacific Exploring Expedition under Captains Ringgold and Rodgers. Proc. Boston Soc. Nat. Hist. 8 (April) 33-40] is one of the most important and best-known oysters in China. Based on the color of its flesh, two forms of C rivularis are recognized and referred to as the "white meat" and 11 red meat" oysters. The classification of white and red forms of this species has been a subject of confusion and debate in China. To clarify the taxonomic status of the two forms of C. rivularis, we collected and analyzed oysters from five locations along China's coast using both morphological characters and DNA sequences from mitochondrial 16S rRNA and cytochrome oxidase 1, and the nuclear 28S rRNA genes. Oysters were classified as white or red forms according to their morphological characteristics and then subjected to DNA sequencing. Both morphological and DNA sequence data suggest that the red and white oysters are two separate species. Phylogenetic analysis of DNA sequences obtained in this study and existing sequences of reference species show that the red oyster is the same species as C. ariakensis Wakiya [1929. Japanese food oysters. Jpn. J. Zool. 2, 359-367.], albeit the red oysters from north and south China are genetically distinctive. The white oyster is the same species as a newly described species from Hong Kong, C. hongkongensis Lam and Morton [2003. Mitochondrial DNA and identification of a new species of Crassostrea (Bivalvia: Ostreidae) cultured for centuries in the Pearl River Delta, Hong Kong, China. Aqua. 228, 1-13]. Although the name C. rivularis has seniority over C. ariakensis and C. hongkongensis, the original description of Ostrea rivularis by Gould [1861] does not fit shell characteristics of either the red or the white oysters. We propose that the name of C. rivularis Gould [1861] should be suspended, the red oyster should take the name C. ariakensis, and the white oyster should take the name C. hongkongensis. (C) 2004 Elsevier B.V. All rights reserved.

Jiangella gansuensis gen. nov., sp nov., a novel actinomycete from a desert soil in north-west China

A novel actinomycete strain, designated YIM 002(T), was isolated from a desert soil sample in Gansu Province, north-west China. This actinomycete isolate formed well-differentiated aerial and substrate mycelia. In the early stages of growth, the substrate mycelia fragmented into short or elongated rods. Chemotaxonomically, it contained LL-2,6-diaminopimelic acid in the cell wall. The cell-wall sugars contained ribose and glucose. Phospholipids present were phosphatidylinositol mannosides, phosphatidylinositol and diphosphatidylglycerol. MK-9(H-4) was the predominant menaquinone. The major fatty acids were anteiso C-15:0 (35.92%), anteiso C-17:0 (15.84%), iso C-15:0 (10.40%), iso C-16:0 (7.07%) and C(17:10)w8c (9.37%). The G+C content of the DNA was 70 mol%. Phylogenetic analysis and signature nucleotide data based on 16S rRNA gene sequences showed that strain YIM 002(T) is distinct from all recognized genera of the family Nocardioidaceae in the suborder Propionibacterineae. On the basis of the phenotypic and genotypic characteristics, it is proposed that isolate YIM 002(T) be classified as a novel species in a new genus, Jiangella gansuensis gen. nov., sp. nov. The type strain is YIM 002(T) (= DSM 44835(T) = CCTCC AA 204001(T) = KCTC 19044(T)).

淡水湖泊中类蛭弧菌的分离鉴定与多样性分析

类蛭弧菌(BALOs)是一种微小的，能够高速运动的革兰氏阴性捕食细菌。它通过裂解宿主细胞来获得自己生长发育所需的物质、能量。类蛭弧菌分布范围广泛，土壤、海洋、湖泊、河流、下水道的污水、水管和水池之中都有它的存在。目前将与蛭弧菌具有相似生活特性的一类细菌称为 “类蛭弧菌”。 该研究从阿哈湖、百花湖和滇池三个内陆淡水湖泊中分离鉴定了宿主菌。利用分离得出的两株宿主菌分离培养类蛭弧菌。进行了16S rRNA基因序列分析。对不同富营养化的高原湖泊中类蛭弧菌的多样性进行比较。在以下几个方面取得了一些进展： 1. 改进了对类蛭弧菌的分离培养方法。我们直接从类蛭弧菌生活的环境中去寻找并提取宿主细菌。这样可以最大程度的保证实验室条件下类蛭弧菌的生长和真实环境中的相似性。我们的实验也证明了这种方法的有效性，并且在实验中我们获得了肠杆菌和黄色假单胞菌这两种类蛭弧菌的宿主菌。 2. 用新分离出来的两种宿主菌，通过噬菌斑生成、吸光度检测，PCR扩增等一系列实验证明我们成功的从淡水湖泊中分离得到了类蛭弧菌。 3、经过对所得的类蛭弧菌的16S rRNA基因序列进行检测,获得了不同类群的类蛭弧菌，并且还发现了新的类群。 3. 构建了分离的类蛭弧菌的系统发生树，对三个湖泊中类蛭弧菌的多样性进行了分析。

Using the ribosomal internal transcribed spacer (ITS) as a complement marker for species identification of red macroalgae

Barcodes based on mitochondrial cytochrome oxidase (mtDNA CO1) sequences are being used for broad taxonomic groups of animals with demonstrated success in species identification and cryptic species discovery, but it has become clear that complementation by a nuclear marker system is necessary, in particular for the barcoding of plants. Here, we propose the nuclear internal transcribed spacer (ITS) as a potentially usable and complementary marker for species identification of red macroalgae, as well as present a primary workflow for species barcoding. Data show that for most red macroalgal genera (except members of the family Delesseriaceae), the size of ITS region ranges from 600 to 1200 bp, and contains enough variation to generate unique identifiers at either the species or genus levels. Consistent with previous studies, we found that the ITS sequence can resolve closely related species with the same fidelity as mtDNA CO1. Significantly, we confirmed that length polymorphism in the ITS region (including 5.8S rRNA gene) can be utilized as a character to discriminate red macroalgal species. As a complementary marker, the verifiable nuclear ITS region can speed routine identification and the detection of species, advance ecological and taxonomic inquiry, and permit rapid and accurate analysis of red macroalgae.