Identification of bacteria associated with Dinoflagellates (Dinophyceae) Alexandrium spp. using tyramide signal amplification fluorescent in situ hybridization and confocal microscopy

In the marine environment, phytoplankton and bacterioplankton can be physically associated. Such association has recently been hypothesized to be involved in the toxicity of the dinoflagellate genus Alexandrium. However, the methods, which have been used so far to identify, localize, and quantify bacteria associated with phytoplankton, are either destructive, time consuming, or lack precision. In the present study we combined tyramide signal amplification–fluorescent in situ hybridization (TSA-FISH) with confocal microscopy to determine the physical association of dinoflagellate cells with bacteria. Dinoflagellate attached microflora was successfully identified with TSA-FISH, whereas FISH using monolabeled probes failed to detect bacteria, because of the dinoflagellate autofluorescence. Bacteria attached to entire dinoflagellates were further localized and distinguished from those attached to empty theca, by using calcofluor and DAPI, two fluorochromes that stain dinoflagellate theca and DNA, respectively. The contribution of specific bacterial taxa of attached microflora was assessed by double hybridization. Endocytoplasmic and endonuclear bacteria were successfully identified in the nonthecate dinoflagellate Gyrodinium instriatum. In contrast, intracellular bacteria were not observed in either toxic or nontoxic strains of Alexandrium spp. Finally, the method was successfully tested on natural phytoplankton assemblages, suggesting that this combination of techniques could prove a useful tool for the simultaneous identification, localization, and quantification of bacteria physically associated with dinoflagellates and more generally with phytoplankton.

(Table S2) Cell concentrations of individual and combined Alexandrium spp. and phylogenetic groups as obtained by qPCR assay and Utermöhl counts, as well as particulate PSP toxin concentrations from discrete water depths

Molecular methods provide promising tools for routine detection and quantification of toxic microalgae in plankton samples. To this end, novel TaqMan minor groove binding probes and primers targeting the small (SSU) or large (LSU) ribosomal subunit (rRNA) were developed for two species of the marine dinoflagellate genus Alexandrium (A. minutum, A. tamutum) and for three groups/ribotypes of the A. tamarense species complex: Group I/North American (NA), Group II/Mediterranean (ME) and Group III/Western European (WE). Primers and probes for real-time quantitative PCR (qPCR) were species-specific and highly efficient when tested in qPCR assays for cross-validation with pure DNA from cultured Alexandrium strains. Suitability of the qPCR assays as molecular tools for the detection and estimation of relative cell abundances of Alexandrium species and groups was evaluated from samples of natural plankton assemblages along the Scottish east coast. The results were compared with inverted microscope cell counts (Utermöhl technique) of Alexandrium spp. and associated paralytic shellfish poisoning (PSP) toxin concentrations. The qPCR assays indicated that A. tamarense (Group I) and A. tamutum were the most abundant Alexandrium taxa and both were highly positively correlated with PSP toxin content of plankton samples. Cells of A. tamarense (Group III) were present at nearly all stations but in low abundance. Alexandrium minutum and A. tamarense (Group II) cells were not detected in any of the samples, thereby arguing for their absence from the specific North Sea region, at least at the time of the survey. The sympatric occurrence of A. tamarense Group I and Group III gives further support to the hypothesis that the groups/ribotypes of the A. tamarense species complex are cryptic species rather than variants belonging to the same species.

Experimental study on the impact of dinoflagellate Alexandrium species on populations of the rotifer Brachionus plicatilis

To investigate harmful effects of the dinoflagellate Alexandrium species on microzooplankton, the rotifer Brachionus plicatilis was chosen as an assay species, and tested with 10 strains of Alexandrium including one known non-PSP-producer (Alexandrium tamarense, AT-6). HPLC analysis confirmed the PSP-content of the various strains: Alexandrium lusitanicum, Alexandrium minutum and Alexandrium tamarense (ATHK, AT5-1, AT5-3, ATC102, ATC103) used in the experiment were PSP-producers. No PSP toxins were detected in the strains Alexandrium sp1, Alexandrium sp2. Exposing rotifer populations to the densities of 2000 cells ml(-1) of each of these 10 Alexandrium strains revealed that the (non-PSP) A. tarnarense (AT-6) and two other PSP-producing algae: A. lusitanicum, A. minutum, did not appear to adversely impact rotifer populations. Rotifers exposed to these three strains were able to maintain their population numbers, and in some cases, increase them. Although some increases in rotifer population growth following exposures to these three algal species were noted, the rate was less than for the non-exposed control rotifer groups. In contrast, the remaining seven algal strains (A. tamarense ATHK, AT5-1, AT5-3, ATC102, ATC103; also Alexandrium sp1 and Alexandrium sp2) all have adverse effects on the rotifers. Dosing rotifers with respective algal cell densities of 2000 cells ml-1 each, for Alexandrium spl, Alexandrium sp2, and A. tamarense strains ATHK and ATC103 showed mean lethal time (LT50) on rotifer populations of 21, 28, 29, and 36h, respectively. The remaining three species (A. tamarense strains AT5-1, AT5-3, ATC102) caused respective mean rotifer LT50S of 56, 56, and 71 h, compared to 160 h for the unexposed "starved control" rotifers. Experiments to determine ingestion rates for the rotifers, based on changes in their Chlorophyll a content, showed that the rotifers could feed on A. lusitanicum, A. minutum and A. tamarense strain AT-6, but could graze to little or no extent upon algal cells of the other seven strains. The effects on rotifers exposed to different cell densities, fractions, and growth phases of A. tamarense algal culture were respectively compared. It was found that only the whole algal cells had lethal effects, with strongest impact being shown by the early exponential growth phase of A. tamarense. The results indicate that some toxic mechanism(s), other than PSP and present in whole algal cells, might be responsible for the adverse effects on the exposed rotifers. (C) 2004 Elsevier B.V. All rights reserved.

Exposure to toxic Alexandrium minutum activates the detoxifying and antioxidant systems in gills of the oyster Crassostrea gigas

Harmful algal blooms of Alexandrium spp. dinoflagellates regularly occur in French coastal waters contaminating shellfish. Studies have demonstrated that toxic Alexandrium spp. disrupt behavioural and physiological processes in marine filter-feeders, but molecular modifications triggered by phycotoxins are less well understood. This study analyzed the mRNA levels of 7 genes encoding antioxidant/detoxifying enzymes in gills of Pacific oysters (Crassostrea gigas) exposed to a cultured, toxic strain of A. minutum, a producer of paralytic shellfish toxins (PST) or fed Tisochrysis lutea (T. lutea, formerly Isochrysis sp., clone Tahitian (T. iso)), a non-toxic control diet, in four repeated experiments. Transcript levels of sigma-class glutathione S-transferase (GST), glutathione reductase (GR) and ferritin (Fer) were significantly higher in oysters exposed to A. minutum compared to oysters fed T. lutea. The detoxification pathway based upon glutathione (GSH)-conjugation of toxic compounds (phase II) is likely activated, and catalyzed by GST. This system appeared to be activated in gills probably for the detoxification of PST and/or extra-cellular compounds, produced by A. minutum. GST, GR and Fer can also contribute to antioxidant functions to prevent cellular damage from increased reactive oxygen species (ROS) originating either from A. minutum cells directly, from oyster hemocytes during immune response, or from other gill cells as by-products of detoxification.

亚历山大藻分子鉴定和荧光原位杂交检测方法研究

近年来，世界沿海国家有害赤潮发生的频率、规模及危害都有上升趋势，有害赤潮已经成为重要的近海环境问题之一。要有效防范有害赤潮带来的危害效应，建立和发展可靠、有效的赤潮监测手段非常重要。目前，对于赤潮藻种的监测主要依靠显微观察的方法，在实际应用中经常遇到困难。首先，亲缘关系相近的物种在形态上差异很小，如甲藻门亚历山大藻属的一些种类，仅细胞壁上个别甲片的结构有细微差别，并且这些形态学指标还容易受环境条件及生长阶段的影响。另外，这种以形态学为基础的分析方法，分析速度慢、耗时长，对操作人员的要求较高，难以满足浮游植物种群动力学监测“量大、连续”的要求。因此，本研究将分子生物学的技术和方法应用于赤潮监测，力求提高赤潮藻种鉴定的准确性和检测工作的效率。 亚历山大藻是一类重要的有害赤潮藻，该藻属中一些产毒特性差别很大的藻种，单从表形特征难以明确区分，从而限制了基于形态观察的监测技术的应用。本研究中，我们尝试应用分子生物学技术与方法，开展了该藻属藻种分子鉴定和荧光原位杂交检测方法的研究。在亚历山大藻的分子鉴定方面，我们采用了核糖体RNA基因（rDNA）序列分析的方法，首次测定了9株分离自中国沿海的（以及实验室保有的其它两株）亚历山大藻的rDNA序列全长，其中包括核糖体小亚基（SSU）rDNA、大亚基（LSU）rDNA、5.8S rDNA及内转录间隔区（ITS）区序列。序列分析结果显示，这些藻株包含了5种核糖体类型，分别是塔玛复合种亚洲温带（Temperate Asian）核糖体类型（TSC-TA），塔玛复合种西欧（West European）核糖体类型（TSC-WE），相关亚历山大藻（A. affine）核糖体类型（AF），微小亚历山大藻（A. minutum）葡萄牙（Portugal）核糖体类型（M-PO）和微小亚历山大藻新西兰（New Zealand）核糖体类型（M-NZ）。将测获的rDNA序列划分为若干保守性不同的区段，分别进行系统发育分析（结合GenBank数据库中保存的其它亚历山大藻相关序列）。结果显示，LSU rDNA D1-D2区是对该藻属藻种进行分子鉴定和系统发育研究的较好区段。同时，为解决建立亚历山大藻克隆培养的困难，我们应用单细胞rDNA序列分析方法，对亚历山大藻单个细胞直接进行了种类鉴定。结果表明，该方法适用于不同生活史阶段的亚历山大藻。 在亚历山大藻的检测技术方面，我们进一步扩展和完善了针对完整细胞的荧光原位杂交检测方法。首先，通过对不同核糖体类型藻株rDNA序列信息的对比分析，针对各自特异的序列位点，设计了特异性rRNA标记探针。经荧光原位杂交实验检验，实现了对5种核糖体类型亚历山大藻的特异性标记。其中，针对WE、M-PO及M-NZ核糖体型的特异性探针为首次获得，另外两个探针是针对TA和AF核糖体类型rRNA新的位点所设计。同时，对影响探针标记效果的诸多因素进行了分析和探讨。此外，在2007年春季长江口海域赤潮调查中，首次应用特异性核酸探针和荧光原位杂交检测方法，调查了该海域亚历山大藻的丰度。结果表明，在4月4日-4月10日的样品中，亚历山大藻达到了较高的密度，最高密度达到103cells/L。同时发现，实验中样品的保存方法有待改进。随后的研究表明，盐醇固定方法及多聚甲醛/甲醇固定方法，可以较好的保持rRNA不被降解并适宜杂交（至少3个月时间）。 总之，本研究首次测定并分析了11株亚历山大藻（9株分离自中国沿海）的rDNA全序列信息。在此基础上，获得了5种核糖体类型亚历山大藻的特异性rRNA标记探针，其中3种为首次获得。另外，实验证明，单细胞rDNA分析技术和荧光原位杂交检测方法，在自然水体中亚历山大藻的直接鉴定及丰度调查中，均具有良好的应用前景。这一工作为我国近海亚历山大藻的鉴定和检测提供了理论依据和方法学基础，希望对该藻赤潮的监测工作有推动作用。 关键词：亚历山大藻 遗传探针 rRNA rDNA 荧光原位杂交 系统发育

Development and validation of an ultrasensitive fluorescence planar waveguide biosensor for the detection of paralytic shellfish toxins in marine algae

Marine dinoflagellates of the genera Alexandrium are well known producers of the potent neurotoxic paralytic shellfish toxins that can enter the food web and ultimately present a serious risk to public health in addition to causing huge economic losses. Direct coastal monitoring of Alexandrium spp. can provide early warning of potential shellfish contamination and risks to consumers and so a rapid, sensitive, portable and easy-to-use assay has been developed for this purpose using an innovative planar waveguide device. The disposable planar waveguide is comprised of a transparent substrate onto which an array of toxin-protein conjugates is deposited, assembled in a cartridge allowing the introduction of sample, and detection reagents. The competitive assay format uses a high affinity antibody to paralytic shellfish toxins with a detection signal generated via a fluorescently labelled secondary antibody. The waveguide cartridge is analysed by a simple reader device and results are displayed on a laptop computer. Assay speed has been optimised to enable measurement within 15 min. A rapid, portable sample preparation technique was developed for Alexandrium spp. in seawater to ensure analysis was completed within a short period of time. The assay was validated and the LOD and CCß were determined as 12 pg/mL and 20 pg/mL respectively with an intra-assay CV of 11.3% at the CCß and an average recovery of 106%. The highly innovative assay was proven to accurately detect toxin presence in algae sampled from the US and European waters at an unprecedented cell density of 10 cells/L. © 2012 Elsevier B.V. All rights reserved.

Novel hydrolysis-probe based qPCR assay to detect saxitoxin transcripts of dinoflagellates in environmental samples

Paralytic Shellfish Poisoning (PSP) is a serious human illness caused by ingestion of seafood enriched with paralytic shellfish toxins (PSTs). PSTs are neurotoxic compounds produced by marine dinoflagellates, specifically by Alexandrium spp., Gymnodinium catenatum and Pyrodinium bahamense. Every year, massive monitoring of PSTs and their producers is undertaken worldwide to avoid PSP incidences. Here we developed a sensitive, hydrolysis probe-based quantitative PCR (qPCR) assay to detect a gene essential for PST synthesis across different dinoflagellate species and genera and tested it on cDNA generated from environmental samples spiked with Alexandrium minutum or Alexandrium fundyense cells. The assay was then applied to two environmental sample series from Norway and Spain and the results were complemented with cell counts, LSU-based microarray data and toxin measurements (enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) biosensor method). The overall agreement between the results of the qPCR assay and the complementary data was good. The assay reliably detected sxtA transcripts from Alexandrium spp. and G. catenatum, even though Alexandrium spp. cell concentrations were mostly so low that they could not be quantified microscopically. Agreement between the novel assay and toxin measurements or cell counts was generally good; the few inconsistencies observed were most likely due to disparate residence times of sxtA transcripts and PSTs in seawater, or, in the case of cell counts, to dissimilar sxtA4 transcript numbers per cell in different dinoflagellate strains or species. © 2013 Elsevier B.V.