Molecular characterization and morphology of the photosynthetic dinoflagellate Bysmatrum caponii from two solar saltons in western Korea

Species belonging to the genus Bysmatrum are peridinoid, thecate, photosynthetic dinoflagellates. The plate formula of Bysmatrum spp., arranged in a Kofoidian series, is almost identical to that of Scrippsiella spp. Bysmatrum spp., which were originally classified as Scrippsiella spp., but were transferred to the genus Bysmatrum spp. because of separation of the intercalary plates 2a and 3a by plate 3??. Whether this transfer from Scrippsiella spp. to Bysmatrum spp. is reasonable should be genetically confirmed. Dinoflagellates were isolated from 2 solar saltons located in western Korea in 2009?C2010 and 3 clonal cultures from Sooseong solar saltons and 2 clonal cultures from Garolim solar saltons were successfully established. All of these dinoflagellates were identified as Bysmatrum caponii based on morphology analysis by light and electron microscopy. The plates of all Korean strains of B. caponii were arranged in a Kofoidian series of Po, X, 4??, 3a, 7??, 6c, 4s, 5?, 0 (p), and 24??. When properly aligned, the ribosomal DNA (rDNA) sequences of the 3 Sooseong strains of B. caponii were identical, as were those of the 2 Garolim strains. Furthermore, the sequences of the 3 Sooseong strains were 0.01% different from those of the Garolim strains. However, the sequences of SSU rDNA of these Korean B. caponii strains were 9% different from that of Bysmatrum subsalsum and > 10% from that of any other dinoflagellate thus far reported. In the phylogenetic trees generated using SSU and LSU rDNA sequences, these Korean B. caponii strains formed a clade with B. subsalsum which was clearly divergent from the Scrippsiella clade. However, this Bysmatrum clade was phylogenetically close to the Protoperidinium and/or Peridinium clades. The results of the present study suggest that Bysmatrum spp. are markedly different genetically from Scrippsiella spp..     

Molecular analysis of complete ssu to lsu rdna sequence in the harmful dinoflagellatealexandrium tamarense (korean isolate, HY970328M)

New PCR primers (N=18) were designed for the isolation of complete SSU to LSU rDNA sequences from the dinoflagellateAlexandrium tamarense. Standard PCR, employing each primer set selected for amplifications of less than 1.5 kb, successfully amplified the expected rDNA regions of A. tamarense (Korean isolate, HY970328M). Complete SSU, LSU rDNAs and ITS sequences, including 5.8S rDNA, were recorded at 1,800 bp, 520 bp and 3,393 bp, respectively. The LSU rDNA sequence was the first report inAlexandrium genus. No intron was found in the LSU rRNA coding region. Twelve D-domains within the LSU rDNA were put together into 1,879 bp (44.4% G+C), and cores into 1514 bp (42.8% G+C). The core sequence was significantly different (0.0867 of genetic distance, 91% sequence similarity) in comparison withProrocentrum micans (GenBank access. no. X16108). The D2 region was the longest in length (300 bp) and highly variable among the 12 D-domains. In a phylogenetic analysis using complete LSU rDNA sequences of a variety of phytoplankton,A tamarense was clearly separated with high resolution against other species. The result suggests that the sequence may resolve the taxonomic ambiguities ofAlexandrium genus, particularly of the tamarensis complex.     

北部湾涠洲岛红色赤潮藻的分子鉴定

为了弄清北部湾涠洲岛6株疑似红色赤潮藻（Akashiwo sanguinea）株的种类,作者采用光学显微镜和荧光显微镜对其进行形态学初步鉴定;并对藻株SSU rDNA、LSU rDNA和ITS序列测序,利用最大似然法构建系统发育树。结果表明,北部湾涠洲岛6株藻与红色赤潮藻形态特征基本符合;3种序列系统发育树分析均发现北部湾涠洲岛6株藻与不同海域来源的红色赤潮藻聚在同一大分支上,自展值高达99、100、100;与来自亚洲海域的韩国安山株、新加坡株、中国厦门港株序列差异最小,亲缘关系最近,而与其他地理来源的红色赤潮藻序列差异大,亲缘关系较远。红色赤潮藻SSU、LSU、ITS种内遗传距离分别为0.001~0.008、0.003~0.025和0.045~0.406,明显小于该藻与其他裸甲藻科（Gymnodiniaceae）藻属下的种间遗传距离0.032~0.072、0.165~0.222和0.589~1.559,因此可确定北部湾涠洲岛6株藻均为红色赤潮藻。研究结果将为北部湾海域赤潮生物来源与组成、赤潮的发生与管理提供基础信息。     

二种核糖体型的多环马格里夫藻(Margalefidinium polykrikoides)的形态和超微结构比较及分子系统学分析

多环马格里夫藻是一种世界性广泛分布的有毒有害藻华原因种。过去30多年里多环马格里夫藻藻华在许多国家频繁暴发且呈快速扩散趋势。过去的研究根据不同地理来源种群在核糖体大亚基基因序列的显著差别,将其分成4种不同核糖体型(ribotype),即东亚型、菲律宾型、美国/马来西亚型和地中海型。但是,不同核糖体型的种群在形态和超微结构上是否也存在可辨识差异尚未见深入研究。为此,本文对从中国青岛胶州湾、美国东海岸和马来西亚近海分离的三株多环马格里夫藻(中国株MPJZBC3、美国株CP1和马来西亚株MPCoKK23)的细胞表面形态和超微结构进行了比较研究,并进一步基于核糖体大亚基序列进行了分子系统学分析。结果首先证明了分离自胶州湾的多环马格里夫藻属于东亚核糖体型,而美国株和马来西亚株均属美国/马来西亚型(中国株与美国株及马来西亚株在1523个碱基的核糖体大亚基序列上相似性为93.6%)。但是,在所有常用的具有分类学意义的细胞形态学和超微结构特征上[细胞大小、单细胞或群体及群体细胞数、上下锥形状、横沟位移度(绕细胞圈数)、纵沟形态、顶沟形态、细胞核位置与形状、染色体形状与数目、叶绿体的形状、结构、数目及分布、淀粉核形态等],中国株与美国株和马来西亚株(也即东亚核糖体型和美国/马来西亚核糖体型)都没有明显可区分的差别。因此,我们认为应该在生活史、休眠孢囊形态和生理学特征(如毒素种类及毒性强弱)以及性别相容性(sexualcompatibility)等方面开展进一步比较研究,以获得对不同核糖体型种群的识别性状。在现阶段有害藻华的野外监测中,尤其是对于不同核糖体型种群可能造成的生物入侵的监测和追溯,多环马格里夫藻的鉴定尚必须以核糖体基因序列作为可靠的判决依据。     

利用rDNA和ITS序列对1株裸甲藻的初步鉴定

测定了 1株分离自青岛胶州湾海水样品、从形态上初步确定为裸甲藻属 (Gymnodiniumsp,编号为 GYN- 1 5)的核糖体基因 (r DNA)和转录单元内间隔区 (Internal Transcribed Spacer,ITS)序列 ,并利用该序列对该藻进行了初步鉴定。测定的序列长 2 658bp,涵盖了小亚基 (smallsubunit,SSU) r DNA基因 3'端 1 747bp,ITS1 - 5.8S r DNA- ITS2全长和大亚基 (large subunit,LSU) r DNA基因 5'端 337bp。同源性分析该序列发现 GYN- 1 5与共生甲藻属 (Symbiodinium)中的 2个种 (Symbiodinium californium和 Gymnodinium varians)的对应序列具有很高的相似性 ;3株藻的各段 r DNA和 ITS序列的相似性均为 99%以上 ;以 SSU r DNA序列中的 3个可变区 (V1 V2 V3)和邻接法构建的系统树表明 ,GYN- 1 5与 S.californium和 G.varians构成 1个独立的新的子类群 ,该子类群属于共生甲藻属 ,而与各种裸甲藻的亲缘关系较远。根据这些结果可将GYN- 1 5初步鉴定为属于共生甲藻属。鉴于 GYN- 1 5和其它 2个种所构成的分支明显与 5个已知的子类群 (A,B,C,D,E)不同 ,因此将该分支命名为子类群 F。     

一株分离自胶州湾的裸甲藻形态相似种的分子系统学研究

对一株分离自胶州湾的裸甲藻形态相似种(Gymnodinium sp.ZX)进行了分子水平的分类鉴定.提取基因组DNA后扩增核糖体小亚基和转录间隔区序列,经纯化、克隆并测序.将获得的序列分别进行Blastn同源性分析,并下载相关序列构建系统进化树,结果表明,该藻与共生藻(Symbiodinium)亲缘关系较近,而与裸甲藻...     

象山港养殖大黄鱼寄生新贝尼登虫成虫形态学和28S rDNA,ITS1分子鉴定

贝尼登类单殖吸虫是象山港海水养殖鱼类中一类危害严重的寄生虫。应用PCR扩增及DNA序列分析的分子生物学手段,并结合对成虫的形态学分析,对象山港养殖大黄鱼体表寄生的贝尼登类单殖吸虫(记作XSp)进行了种类鉴定,结果表明XSp从形态特征上属于新贝尼登虫属,与梅氏新贝尼登虫高度相似。扩增得到XSp的28S rDNA和ITS1序列长度分别为393和427 bp,与梅氏新贝尼登虫和鱾新贝尼登虫的5个28S rDNA 序列、2个ITS1序列的比对分析显示相似性除1个为97.4%外其余均大于99%,提示XSp与这几个鱾新贝尼登虫和梅氏新贝尼登虫为种内关系,而XSp与贝尼登虫的3个28S rDNA 和1个ITS1序列相似性分别为84.3%~89.5%和60.2%。系统进化树显示该吸虫与梅氏新贝尼登虫和鱾新贝尼登虫形成一个紧密的簇,而与贝尼登虫亲缘关系较远。根据普通生物学和序列特征分析,将XSp定种为梅氏新贝尼登虫,并且支持Whittington和Horton (1996)提出的梅氏新贝尼登虫和鱾新贝尼登虫为同种异名的分类观点。     

新月细柱藻的分离培养及形态和分子鉴定

自胶州湾分离培养2株微藻(MGB0401和MGB0402)。经形态学鉴定为新月细柱藻(Cylindrotheca closterium)。MGB0401和MGB0402的18S核糖体RNA基因(rDNA)扩增片段长度为1775 bp,存在10个碱基差异。MGB0401和MGB0402的rbcL基因扩增片段长度为1190 bp,有45个碱基差异,对应的氨基酸序列存在4个氨基酸残基差异。系统学分析表明,2株微藻与已知新月细柱藻相应序列亲缘关系最近。微藻常用系统学分析用基因的序列不仅是分类鉴定的补充指标,也是不同研究结果相互比较的重要基础。     

Assessment of introduced Kappaphycus (Solieriaceae, Rhodophyta) species relationships in China with molecular markers

Due to morphological plasticity and paucity of diagnostic morphological characters, the taxonomy of Kappaphycus gets more and more confused with the expanding of commercial cultivation. In this study, the phylogenetic relationship of 13 strains of introduced Kappaphycus species in China was defined using DNA molecular markers, such as 18S rDNA, rbcL and cox2-cox3 spacer region. The resolutions obtained by three different molecular markers were compared: both cox2-cox3 spacer region and rbcL sequences are eligible in interspecies identification of Kappaphycus, whereas cox2-cox3 spacer region is more variable than rbcL sequence. There is several basepairs’ discrepancy among 18S rDNA sequences, while it is 100% identical among both cox2-cox3 spacer region and rbcL sequences of the ten strains of K. alvarezii. We suppose that 18S rDNA sequence can provide more information in biogeography study of Kappaphycus than other two DNA sequences.     

Molecular identification of bloom-forming species Phaeocystis globosa (Pryninesiophyta) and its dispersal based on rDNA ITS sequence analysis

The colony-forming Phaeocystis species are causative agents of dense bloom occurrences in coastal waters worldwide. It is difficult to separate them because of the different morphologies associated with their colonial stages. In this study we applied molecular approaches to analyze the genetic variation of Phaeocystis globosa and Phaeocystis pouchetii from several geographic regions, and to assist in tracing the dispersal of bloom-forming Phaeocystis species in coastal waters of China. The sequences of the internal transcribed spacers (ITS1 and ITS2) of rDNA and the 5.8S ribosomal RNA gene of Phaeocystis strains were determined. Sequence comparison shows that P.globosa was the most divergent to P. pouchetii, exhibiting sequence divergence higher than 0.08. However, lower genetic divergences existed between strains of P.globosa. The sequence comparison of the Phaeocystis rDNA ITS clearly shows that the species isolated from the southeast coast of China is identified as P.globosa rather than P. cf. pouchetii or other species. Furthermore, the significance of rDNA variation in distinct global populations of P.globosa suggested it might have had sufficient time to accumulate detectable mutations at the rDNA locus, supporting the hypothesis of ancient dispersal of P.globosa to many areas, meaning that P.globosa blooms in the coastal waters of China are endemic rather than a newly introduced species or a foreign source. Finally, based on the high divergent region of rDNA ITS, a pair of species-specific primers for P.globosa were designed, they could be useful to detect the presence of this species in mixed plankton assemblages or flagellate stages that are recognized with diffculties by means of conventional microscopy.     

分离自中国胶州湾的褐色马格里夫藻(Margalefidinium fulvescens)的形态学和系统进化特征

The dinoflagellate genus Margalefidinium has been split from Cochlodinium as a new genus recently and Margalefidinium fulvescens is one of the five Margalefidinium species. Margalefidinium fulvescens is toxic and has been reported from the coastal waters of USA, Canada, Mexico, China, Japan, Indonesia, Korea, Pakistan and Spain. Here we provide the morphological and phylogenetic characterization for an isolate of it from the Jiaozhou Bay, Qingdao, China. Our results showed that the vegetative cells were subspherical to ellipsoidal, 34–60 μm in length, and 19–41 μm in width. Both single cell forms and colonies in chains of 2, 4, or 8 cells were observed in cultures, but chain forms with 2 or 4 cells were observed more often in the field samples. The cingulum was rather deep, encircling the cell approximately twice, but the sulcus was rather narrow, surrounding the cell about one turn. The nucleus was spherical and located at the central epicone. The chloroplasts were granular, brownish, and scattered peripherally. An orange pigmented body also appeared in the epicone. The apical groove appeared vase-like as previously described. Under epi-fluorescence microscopy, a pumpkin-like structure was clearly observed, in which cells were embedded. Cells were observed to exit from the structure, which led us to a hypothesis that the structure may provide cells a shelter to avoid predation or to respond to other stresses. The phylogenetic analyses based on partial LSU rDNA sequences indicated that M. fulvescens from the Jiaozhou Bay was grouped with M. fulvescens populations from other origins and closely related to the clade of M. polykrikoides.Our morphological observations and phylogenetic analyses together confirmed the presence of M. fulvescens in China and our monitoring has also observed the species dominant in the dinoflagellate community of the Jiaozhou Bay in the early autumn of 2015, which alerted us to continually monitor this bloom-forming species in the region.     

Abundance of epiphytic dinoflagellates from coastal waters off Jeju Island,Korea During Autumn 2009

The occurrence of harmful epiphytic dinoflagellates is of concern to scientists, the aquaculture industry, and government due to their toxicity not only to marine organisms but also to humans. There have been no studies on the abundance of the epiphytic dinoflagellates in Korean waters. We explored the presence of epiphytic dinoflagellates in the coastal waters off Jeju Island, southwestern Korea. Furthermore, we measured the abundance of epiphytic dinoflagellates on the thalli of 24 different macroalgae, collected from five different locations in October 2009. Five epiphytic dinoflagellate genera Amphidinium, Coolia, Gambierdiscus, Ostreopsis, and Prorocentrum were found. These five genera were observed on the thalli of the macroalgae Chordaria flagelliformis, Martensia sp., Padina arborescens, and Sargassum sp., while none were observed exceptionally on Codium fragile. The abundance of Ostreopsis spp. was highest on Derbesia sp. (8,660 cells/g wet weight), while that of Gambierdiscus spp. was highest on Martensia sp. (4,870 cells/g-ww). The maximum abundances of Amphidinium spp., Coolia spp., and Prorocentrum spp. were 410, 710, and 300 cells/g-ww, respectively. The maximum abundance of Coolia spp., Gambierdiscus spp., and Ostreopsis spp. obtained in the present study was lower than for other locations reported in literature. The results of the present study suggest that the presence and abundance of epiphytic dinoflagellates may be related to the macroalgal species of the coastal waters of Jeju Island.     

广西北部湾5株骨条藻的分子鉴定

骨条藻(Skeletonema)隶属于硅藻门(Bacillariophyta),是一种分布广泛的广温广盐性藻类,也是我国沿海常见的赤潮生物之一.为确定北部湾涠洲岛5株骨条藻的分类地位,采用PCR克隆了这5株骨条藻核糖体的大亚基基因(LSU)和内转录间隔区(ITS)序列.通过与GenBank数据库中骨条藻属的LSU、IT...     

Combined effects of a parasite,QPX, and the harmful-alga,Prorocentrum minimum on northern quahogs,Mercenaria mercenaria

Northern quahogs, Mercenaria mercenaria (L.), frequently are infected with the parasite Quahog Parasite Unknown (QPX, Labyrintohomorpha, Thraustochytriales), which can cause morbidity and mortality of the quahogs. Possible interactions between this parasitic disease and exposure to the harmful dinoflagellate Prorocentrum minimum in M. mercenaria were studied experimentally. Quahogs from Massachusetts with variable intensity of QPX infection were exposed, under controlled laboratory conditions, to cultured P. minimum added to the natural plankton at a cell density equivalent to a natural bloom. After 5 days of exposure, individual clams were diagnosed histologically to assess prevalence and intensity of parasitic infection, as well as other pathological conditions. Further, cellular defense status of clams was evaluated by analyzing hemocyte parameters (morphological and functional) using flow-cytometry. Exposure of quahogs to P. minimum resulted in: a lower percentage of phagocytic hemocytes, higher production of reactive oxygen species (ROS), larger hemocyte size, more-numerous hemocytic aggregates, and increased numbers of hemocytes in gills accompanied by vacuolation and hyperplasia of the water-tubular epithelial cells of the gills. Quahogs had a low prevalence of QPX; by chance, the parasite was present only in quahogs exposed to P. minimum. Thus, the effect of QPX alone on the hemocyte parameters of quahogs could not be assessed in this experiment, but it was possible to assess different responses of infected versus non-infected quahogs to P. minimum. QPX-infected quahogs exposed to P. minimum had repressed percentage of phagocytic hemocytes, consistent with immuno-modulating effect of P. minimum upon several molluscan species, as well as smaller hemocytes and increased hemocyte infiltration throughout the soft tissues. This experiment demonstrates the importance of considering interactive effects of different factors on the immunology and histopathology of bivalve shellfish, and highlights the importance of considering the presence of parasites when bivalves are subjected to harmful-algal blooms.     

白缘(鱼央)5S rDNA的扩增分析及其在染色体上的FISH定位

本文分别对雌雄白缘(鱼央)的5S rDNA进行了PCR扩增和序列分析,并采用双色荧光原位杂交(FISH)技术,以白缘(鱼央)的5S rDNA序列和小麦的45S rDNA为探针,对其在白缘(鱼央)雌雄中期染色体上进行了FISH定位研究。结果表明,白缘(鱼央)5S rDNA序列无雌雄差异;5S rDNA的保守区序列为117 bp;5S rDNA和45S rDNA分别被定位于白缘(鱼央)的性染色体和第5号染色体上。同时从GenBank中获得了22种鱼的5S rDNA,运用DNAman软件构建了23种鱼的系统发育树,对白缘(鱼央)的进化地位进行了初步研究。本研究为阐明白缘(鱼央)在鱼类系统进化中的位置、重复序列在脊椎动物性染色体上的分布状况以及与性别决定与分化的关系,提供了资料积累和分析依据。     

南海赤潮亚历山大藻的分子鉴定及"塔玛复合种"的分子系统发生分析

利用SSu rDNA和ITS分子指标,结合GenBank和其他文献中的基因序列构建了系统树,对一株分离自我国南海的亚历山大藻"塔玛复合种"NH01进行了鉴定,发现SSU rDNA和ITS序列构建的系统树中,不同地理基因型的"塔玛复合种"均构成独立分支.与我国沿海所有的"塔玛复合种"一致,NH01也属于"亚洲温带"型.探...     

不同养殖区红藻表面假交替单胞菌多样性分析

利用2216E培养基从我国沿海6种养殖红藻的20个样品表面分离了327 株假交替单胞菌。经过16S rDNA序列鉴定, 分别隶属于Pseudoalteromonas carrageenovora、P. haloplanktis、P. marina、P. agarivorans、P. elyakovii和P. lipolytica 6个种。其中P. carrageenovora数量最多, 总共211株, 在14个样品上均有发现。     

北部湾海域球形棕囊藻遗传多样性分析

南海北部湾海域近年来连年暴发球形棕囊藻赤潮,严重威胁近海生态安全,亟待探明赤潮成因。球形棕囊藻是一种广泛分布于温带至热带海域的有害藻华微藻,具有明显的种下遗传分化。为了解北部湾海域球形棕囊藻的遗传多样性及其对赤潮藻种来源的指示意义,本研究针对分离于北部湾海域赤潮发生期间的4株棕囊藻,以核糖体28S大亚基rRNA基因（28S rDNA）D1-D2区和转录间隔区（ITS区）为靶区构建克隆文库,通过测序分析了北部湾海域球形棕囊藻的遗传多样性。结果表明,从北部湾分离的4株棕囊藻均为球形棕囊藻,但不同年份分离的球形棕囊藻藻株之间存在明显的遗传差异。部分球形棕囊藻藻株（PG2015和PG2017）的不同克隆间也存在遗传差异,这种遗传差异在由单一藻细胞建立的克隆培养系中仍存在,表明遗传差异是来自细胞内多拷贝的基因序列,可能源自不同地理种群球形棕囊藻之间的基因交流。与ITS区相比,28S rDNA D1-D2区能更好地反映球形棕囊藻遗传多样性状况,具有作为分子标记指示球形棕囊藻地理种群的潜力,但仍需深入研究。研究结果深化了对北部湾球形棕囊藻遗传多样性的认识,有望为进一步解析北部湾海域球形棕囊藻赤潮原因种来源提供判据。     

Molecular identification of bloom-forming species Phaeocystis globosa(Prymnesiophyta)and its dispersal based on rDNA ITS sequence analysis

Abstract-The colony-forming Phaeocystis species are causative agents of dense bloom occurrences incoastal waters worldwide. It is difficult to separate them because of the different morphologies associatedwith their colonial stages. In this study we applied molecular approaches to analyze the genetic variationof Phaeocystis globosa and Phaeocystis pouchetii from several geogrtaphic regions, and to assist in tracingthe dispersal of bloom-forming Phaeocystis species in coastal waters of China. The Sequences of the inter-nal transcribed sacers (ITS1 and ITS2) of rDNA and the 5.8S ribosomal RNA gene of Phaeocystisstrains were determined. Sequence comparison shows that P.globosa was the most divergent toP. pouchetii, exhibiting sequence divergence higher than 0.08. However, lower genetic divergences ex-isted between strains of P.globosa. The sequence comparison of the Phaeocystis rDNA ITS clearlyshows that the species isolated from the southeast coast of China is identified as P. globosa rather thanP. cf. po