Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced, and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with flu...     

Identification of Provocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified,cloned and sequenced,and these sequence data were deposited in the GenBank.Eight oligonucleotide probes(DNA probes)were designed based on the sequence analysis.The probes were employed to detect and identify P.minimum and T. pulchella in unialgal and mixed algal samples with a fuorescence in situ hybridization method using flow cytometry.Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences,and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe.These DNA probes and the hybridization protocol we developed were specific and effective for P.minimum and T. pulchella,without any specific binding to other algal species.The hyrbridization efficiency of difierent probes specific to P.minimum was in the order:PMl8S02＞PM28S02＞PM28S01＞PM18S01,and that of the probes specific to T. pulchella was TP18S02＞TP28S01＞TP28S02＞TP18S01.The djfferent hybridization efficiency of the DNA probes could also be shown in the fuorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry.The DNA probes PM18S02,PM28S02,TPl8S02 and TP28S01,and the protocol,were also useful for the detection of algae in natural samples.     

应用双特异分子探针技术定性定量检测圆海链藻(Thalassiosira rotula)

设计了一套圆海链藻(Thalassiosira rotula)特异性探针,运用双特异分子探针技术,对圆海链藻(Thalassiosira rotula)进行了定性定量检测.结果表明,本实验中设计的一套探针与其它十几种藻无交叉反应,具有种特异性;细胞裂解液直接杂交检测优于提纯RNA样品检测;对自然样品做了初步检测,发现天然海水中的其它浮游生物对该检测方法影响很小.     

海洋贝类的病害性纤毛虫研究Ⅲ.触毛亚目盾纤类纤毛虫

报道山东沿海贝类鳃表及外套腔中共栖的 3种触毛亚目盾纤类纤毛虫 :厚鱼钩虫(Ancistrum crassum)、日本鱼钩虫 (Ancistrum jap onicum)及亚桶形后口虫 (Boveriasubcylindrica)。文中依据活体观察、蛋白银及银浸法染色对其活体形态学、纤毛图式及银线结构进行了综合描述 ,对采自 4种不同宿主的厚鱼钩虫种群进行了比较研究。该文系后口虫属纤毛虫在我国的首次报道。     

荧光光谱结合主成分分析对硅藻和甲藻的识别测定

将浮游植物三维荧光光谱与主成分分析方法相结合,针对我国东海常见的12种藻建立了硅藻和甲藻的识别测定技术。根据Fisher判别分析结果,选用三维荧光光谱的第一主成分得分谱作为硅藻和甲藻的识别特征谱,建立了浮游植物荧光特征谱库。在此基础上,利用非负最小二乘法进行识别测定,其中单门类样品共有78个,识别正确率为95%,回收率≥81%;2个门类的混合样品共有18个,识别正确率为83%,回收率≥80%。     

Development of taxonomic rRNA-targeted probes of two harmful algae: Prorocentrum minimum and Karenia mikimotoi by fluorescence in situ hybridization

Harmful algal blooms recently have been under the spotlight throughout the world, because of their negative impact on the marine environment, aquaculture, fisheries as well as public health. The development of methods for rapid and precise identification and quantification of causative species is essential for the warning and monitoring of blooms, among which the techniques based on taxonomic probes are the most favored. In this study, two harmful algae, i.e., Prorocentrum minimum and Karenia mikimotoi were taken into consideration. The partial large subunit rDNA (D1-D2) of both species were firstly PCR-amplified, cloned and sequenced. The obtained sequences were then introduced to carry out alignment analysis for gene specific regions. Three respective candidate probes for each species were designed and used to screen the optimal probe by performing fluorescence in situ hybridization (FISH) tests. The results showed that the probes Pmin0443 and Kmik0602 displayed the best hybridization for P. minimum and K. mikimotoi, respectively. Both the specific (taxonomic) (Pmin0443 and Kmik0602) and the control probes (UniC0512 and UniR0499) were used for cross-reactivity tests with other microalgae in our laboratory. The probes Pmin0443 and Kmik0602 are specific and could be served as taxonomic probes introduced into the techniques targeting rRNA, such as FISH, sandwich hybridization, and DNA-microarray assay of P. minimum and K. mikimotoi in the future. Finally, FISH analyses with both probes were performed on the simulated field samples. The probes could hybridize exclusively with the target cells well, and no significant difference (p >0.05) was observed in the cell densities of the samples determined by FISH and light microscopy (LM). All suggest that the probes are specific and could be introduced into FISH for the monitoring of both harmful algae.     

International accreditation of sandwich hybridisation assay format DNA probes for micro‐algae

The sandwich hybridisation assay (SHA) is a DNA probe‐based method for rapid identification and enumeration of toxic micro‐algae which uses species specific oligonucleotide probes targeted at ribosomal RNA. It is suited to fragile micro‐algal cells which commonly collapse during the fixation stage of sample collection, compromising identification by traditional microscopy. The assay has been available for research for several years, but was validated and accepted for international accreditation for commercial laboratory use in New Zealand in May 2004 (International Accreditation New Zealand: ISO 17025). During the validation of the raphidophyte assay, some discrepancies were noted between SHA cell concentration estimates and traditional light microscope cell counts. Higher SHA estimates were recorded when blooms had collapsed but rRNA was still present in sea water. Conversely, higher traditional cell counts occurred when sample delivery was delayed more than 48 h, presumably owing to degradation of rRNA in the live cultures used for the SHA. SHA cell concentration estimates of the toxic diatom bloom‐former Pseudo‐nitzschia australis were also compared with whole cell format DNA probe counts and traditional microscope counts; SHA counts were comparable for the three methods tested.     

杂色鲍鲍苗“掉板症”病因的探讨

针对目前杂色鲍育苗过程中出现的大规模死亡进行了流行病学调查,结果显示,在整个育苗周期内,病害均可发生,死亡率达100%,监测了水质理化因子、原生动物、寄生虫及病原微生物,水质理化因子、原生动物等与鲍苗死亡有一定关系,但不是主要原因;在鲍苗体内分离到的细菌及感染试验结果验证细菌是引起杂色鲍幼苗死亡的主要原因.     

Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified,cloned and sequenced,and these sequence data were deposited in the GenBank.Eight oligonucleotide probes(DNA probes)were designed based on the sequence analysis.The probes were employed to detect and identify P.minimum and T. pulchella in unialgal and mixed algal samples with a fuorescence in situ hybridization method using flow cytometry.Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences,and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe.These DNA probes and the hybridization protocol we developed were specific and effective for P.minimum and T. pulchella,without any specific binding to other algal species.The hyrbridization efficiency of difierent probes specific to P.minimum was in the order:PMl8S02＞PM28S02＞PM28S01＞PM18S01,and that of the probes specific to T. pulchella was TP18S02＞TP28S01＞TP28S02＞TP18S01.The djfferent hybridization efficiency of the DNA probes could also be shown in the fuorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry.The DNA probes PM18S02,PM28S02,TPl8S02 and TP28S01,and the protocol,were also useful for the detection of algae in natural samples.     

Image processing of underwater multispectral imagery

Capturing in situ fluorescence images of marine organisms presents many technical challenges. The effects of the medium, as well as the particles and organisms within it, are intermixed with the desired signal. Methods for extracting and preparing the imagery for analysis are discussed in reference to a novel underwater imaging system called the low-light-level underwater multispectral imaging system (LUMIS). The instrument supports both uni- and multispectral collections, each of which is discussed in the context of an experimental application. In unispectral mode, LUMIS was used to investigate the spatial distribution of phytoplankton. A thin sheet of laser light (532 nm) induced chlorophyll fluorescence in the phytoplankton, which was recorded by LUMIS. Inhomogeneities in the light sheet led to the development of a beam-pattern-correction algorithm. Separating individual phytoplankton cells from a weak background fluorescence field required a two-step procedure consisting of edge detection followed by a series of binary morphological operations. In multispectral mode, LUMIS was used to investigate the bio-assay potential of fluorescent pigments in corals. Problems with the commercial optical-splitting device produced nonlinear distortions in the imagery. A tessellation algorithm, including an automated tie-point-selection procedure, was developed to correct the distortions. Only pixels corresponding to coral polyps were of interest for further analysis. Extraction of these pixels was performed by a dynamic global-thresholding algorithm.     

皱纹盘鲍及其饵料营养成分的研究

分析了不同生长时期的皱纹盘鲍及其天然饵料──硅藻、海带、裙带菜、 石药及１７种人工配合饵料的蛋白质、氨基酸、无机元素粗脂肪等营养成分。明了 不同生长时期的鲍对蛋白质等营养成分的需求量不同，天然饵料和人工配合饵料 之间具有明显差异等。这对根据不同生长时期来选用合适的鲍饵料、人工配合饵料 以符合鲍的营养需求，以及鲍苗食性转化机制等提供了一定的科学依据。     

Species delimitation in the green algal genus Codium (Bryopsidales) from Korea using DNA barcoding

Codium, one of the largest marine green algal genera, is difficult to delimit species boundary accurately based on morphological identification only. DNA barcoding is a powerful tool for discriminating species of seaweeds. The plastid elongation factor TU(tuf A) is considered as maker to perform DNA barcoding of green algal species than rbc L gene due to universality and rapid evolution rate. We conducted DNA barcoding application to Codium specimens from the Jeju Island, Korea to overcome the limit of morphological identification and to confirm the species diversity. As a result of applying tuf A marker, we newly generated fifty-five tuf A barcodes to resolve eight species. Tuf A marker exhibited 6.1%–21.8% interspecific divergences, wider than the gap of rbc L exon 1,3.5%–11.5%. Molecular analysis of rbc L exon 1 sequences of Codium revealed eight distinct species like tuf A analysis separated in five phylogenetic groups. DNA barcoding of the genus Codium using tuf A marker is more helpful to overcome the limit of morphological identification, and this is more potential to reveal cryptic species and to resolve the relationships among subspecies than rbc L analysis alone. The complement of tuf A barcoding and rbc L analyses including morphology for the genus Codium in the northwestern Pacific will give much more reliable achievement for discovering species diversity and resolving the phylogenetic relationships.     

荧光技术在溢油鉴别中的应用

荧光光谱具有灵敏度高、选择性好、分析速度快等特点被广泛应用于溢油鉴别。通过结合同步荧光光谱法、导数荧光光谱法和三维荧光光谱法对某码头一次溢油事故的溢油样进行分析鉴别,快速准确地鉴别出溢油源,进一步说明了荧光光谱法是溢油鉴别中一种有效的技术方法。研究结果表明:(1)同步荧光光谱对油源较为相近的油样区分能力较差,但可初步判断其相似性;(2)三维荧光光谱图油样荧光强度会受风化等因素的影响,但谱图的整体轮廓受干扰因素影响较小,芳烃的测定结果能比较准确地反映出溢油源的特征信息;(3)一阶导数荧光光谱中峰的比值能增强光谱特征,提高溢油鉴别的分辨能力。     

菲律宾蛤仔鳃寄生触毛类纤毛虫——厚鱼钩虫(Ancistrum crasum)的形态学研究(原生动物,纤毛门,盾纤目)

报道了采自青岛胶州湾的菲律宾蛤仔鳃丝上寄生的一种触毛类纤毛虫，经鉴定为厚鱼钩虫（ＡｎｃｉｓｔｒｕｍｃｒａｓｓｕｍＦｅｎｃｈｅｌ，１９６５）。本文运用活体观察和蛋白银法对其活体形态及纤毛图式进行了详细描述。此为我国新记录，同时也是触毛亚目纤毛虫在我国的首次报道。     

Synchronous fluorescence spectrometry of 1-hydroxypyrene: a rapid screening method for identification of PAH exposure in tissue from marine polychaetes

The uptake of polycyclic aromatic hydrocarbons (PAHs) by marine deposit-feeding invertebrates can be determined by screening for PAH-derived metabolites. We identified 1-hydroxypyrene as the only intermediate metabolite in tissue of four species of deposit-feeding polychaetes, Nereis diversicolor, Nereis virens, Arenicola marina, and Capitella sp. I exposed to pyrene spiked sediment. Synchronous fluorescence spectroscopy (SFS) provides a fast and simple method for both qualitative and quantitative analysis of 1-hydroxypyrene in all four species. The SFS assay was validated using HPLC with ultraviolet detection. A good correlation between 1-hydroxypyrene concentrations determined by the two methods was observed. We used HPLC with fluorescence detection combined with enzymatic hydrolysis of conjugated metabolites to investigate species specific metabolite patterns. A tentative aqueous metabolite identification scheme indicates that Nereid polychaetes predominately make use of glucuronide conjugation whereas Capitella sp. I. and Arenicola marina appear to utilize predominantly sulfate and/or glucoside conjugation. The usefulness of 1-hydroxypyrene as a biomarker for PAH exposure in deposit-feeding invertebrates is discussed.     

Horse mackerel egg identification using DNA methodology

Egg identification in plankton samples is usually needed for purposes of stock assessment. Until recently, only morphological characters were used for identifying the eggs of different fish species. Late developmental stages are easily distinguishable due to pigmentation as well as egg and oil globule size range. However, for early stages, these characteristics are difficult to be discriminated and may overlap with other species. European horse mackerel species (Trachurus trachurus, T. mediterraneus, T. picturatus) overlap significantly in their spawning areas in some European waters. Because of the fact that their eggs are morphologically similar, genetic methodologies are needed to identify eggs and larvae to species correctly. In the present study, formalin‐ and ethanol‐preserved eggs were tested to estimate the efficacy of genetic methodologies for species identification of eggs when different preservatives are used. A 370‐bp fragment of cytochrome b was successfully amplified followed by restriction fragment analysis with two restriction enzymes aiming to identify the eggs to species. Horse mackerel egg identification was accomplished with the maximum success in ethanol‐preserved eggs. However, it seems that various preservatives had different effects on the DNA quality of eggs as genetic identification was less successful in formalin‐preserved eggs. Preserving in ethanol a part of the eggs obtained in plankton surveys is suggested for purposes of accurate genetic identification, even if their morphological features are distorted in time.     

基于形态学和宏条形码技术的南海西沙群岛浮游动物多样性的比较分析

西沙群岛是我国南海陆地面积最大的群岛,海域广阔,拥有重要的战略地位与丰富的海洋资源。为了解该海域浮游动物组成与分布特征,本研究于2019年5月在西沙群岛14个岛礁站位开展多学科综合调查,并采用形态学方法和基于18S V9测序的宏条形码技术对浮游动物样本组成进行鉴定。结果显示,此次西沙调查站位浮游动物样本的主要种类包括桡足类、软甲纲和箭虫纲,这3个类别的物种在两种鉴定方法中均具有较高的相对丰度。14个站位浮游动物平均密度为707.53±378.34 ind/m³,各站位浮游动物丰度、物种组成及优势种存在差异。形态学方法共鉴定出11门17纲18目共86个物种,18S V9分子方法鉴定出22门46纲85目共233个操作分类单元(operational taxonomic units, OTUs),分子鉴定的物种覆盖度更高,且代表性类群相对丰度和多样性指数在大部分站位与形态学鉴定结果呈现出显著相关性,表明宏条形码技术鉴定方法与形态学鉴定方法在评价海洋浮游动物多样性方面具有较好的可比性,在我国海洋浮游动物群落监测中具有较高的应用潜力。但由于目前浮游动物的分子鉴定方法仍处于初步发展阶段,相关技术手段仍不完善,仍需多种鉴定方法结合使用,以保证浮游动物多样性鉴定的准确性。     

基于图像识别的5个不同产地克氏原螯虾(Procambarus clarkii)形态差异分析

为了解江苏、江西、湖北、上海、河南5个地区克氏原螯虾（Procambarus clarkii）的形态差异和获取快速、有效的形态鉴别方法,本研究采用传统形态测量法和地标点法来分析各产地形态差异。结果显示：（1）克氏原螯虾雌雄群体相对扭曲主成分分析,前三个主成分累计贡献率分别为79.96%、67.21%,传统形态测量法前三个主成分累计贡献率分别为76.77%、82.70%,两种方法均表明其形态差异主要体现在头胸甲及腹部部位;（2）聚类分析将克氏原螯虾5群体聚为两支,上海、河南、江西、湖北群体聚为一支,江苏群体单独聚为一支。（3）地标点法雌雄群体综合判别准确率分别为100%、94%,传统形态测量法综合判别准确率均为56%。以上研究结果表明不同产地间克氏原螯虾具有一定的形态差异,且地标点法区分不同产地克氏原螯虾群体差异性效果显著,这将有利于克氏原螯虾生产和选育过程中群体的鉴别及外形特征的快速获取。     

北极海洋沉积物中可培养细菌及其多样性分析

利用Zobell 2216E培养基和涂布平板法对北极海洋沉积物中可培养细菌进行分离纯化,并利用16SrRNA基因进行分子鉴定与系统发育分析。根据菌落形态学特征,从59个站点的沉积物样品中共分离纯化获得570株细菌;基于16SrRNA基因的分子鉴定与系统发育分析表明,分离到的可培养细菌分别属于细菌域的4个门,5个纲,12个目,23个科,47个属,102个种,其中γ-Proteobactria占绝大多数;有14株菌株与模式菌株的16SrRNA基因序列相似性小于97%,为6个潜在的新种。北极海域的海洋沉积物中存在着丰富的微生物种质资源,为开发新型生物活性物质和特殊功能基因打下了基础。     

A new method to detect viable bacteria in natural seawater using 16SrRNA oligonucleotide probe

Fluorescent oligonucleotide probes were used to detectVibrio cholerae directly under epifluorescence microscope. The probe which is complementary to the specific 16SrRNA sequence ofVibrio cholerae was labelled with Texas-Red, whereas the universal probe was labelled with Fluorescein. These probes allowed the distinctionVibrio cholerae from other eubacteria under the same microscopic field. In order to detect and enumerate specific bacteria in natural seawater, this method was combined with the direct viable count (DVC) technique. The combined method increased intracellular rRNA levels in the sample, and made it possible to detect the target bacteria with the specific gene probe. The applicability of this new method was confirmed both for the laboratory mixed culture system and natural seawater.