亚历山大藻（非PSP产毒株）毒理的初步研究

本文以一株不产PSP毒素的相关亚历山大藻（AC-1）为对象，研究了该藻株对褶皱臂尾轮虫、卤虫、黑褐新糠虾急性毒性效应和对糠虾的慢性毒性影响，同时对AFT毒素成分进行了研究，研究结果如下： 通过相关亚历山大藻（AC-1）对褶皱臂尾轮虫、卤虫、黑褐新糠虾的急性毒性影响研究，发现随着相关亚历山大藻（AC-1）密度的升高，轮虫、卤虫、糠虾的存活率逐渐降低，其96hLC50分别为：1500cells/ml，90cells/ml，5000cells/ml。比较研究三种生物对相关亚历山大藻（AC-1）敏感性可以看出，三种生物对该藻的敏感性顺序为：卤虫>轮虫>糠虾。 相关亚历山大藻（AC-1）对黑褐新糠虾的生长及种群繁殖有显著影响，我们发现在密度为50cells/ml藻液中，糠虾的繁殖就受到了不利影响。实验进行到63d结束时，糠虾日最高产虾数、总产幼虾数、总产虾天数都明显减少，初次产虾时间推迟，繁殖中断增加。且该藻对黑褐新糠虾亲虾的存活、生长也有一定的影响，糠虾亲虾的存活率为对照的71%，而体长和体重分别为对照组的87.3%和97.8%，但差异尚不显著（P>0.05）。 研究相关亚历山大藻（AC-1）各组分的毒性（藻液、藻细胞重悬液、藻细胞培养过滤液、内容物），发现藻液和藻细胞重悬液对褶皱臂尾轮虫种群数量及轮虫、卤虫、糠虾的存活率均有显著影响，表明相关亚历山大藻活体藻细胞的毒性最强。过滤液和内容物也显著降低了轮虫和卤虫的存活率，其对糠虾也有影响，但不显著，表明AFT毒素可能来源于细胞内，能分泌到细胞培养液中。 研究相关亚历山大藻（AC-1）AFT毒素的热稳定性、酸碱稳定性、去蛋白组分毒性、分子量范围、极性、多糖组分鉴定，表明AFT毒素为极性较强的多糖类物质，对热和酸碱是稳定的，其分子量范围在5K~50K之间。 以上结果表明相关亚历山大藻（AC-1）虽然不产生PSP毒素，但能产生极性多糖类毒素，对甲壳类等浮游动物的种群数量和资源补充产生不利影响。本研究为以后进一步研究AFT毒素的作用机制和毒素的化学结构奠定了基础，为全面评价亚历山大藻赤潮的危害提供了科学依据。

The effect of the causative algae of large-scale HAB in the East China Sea on egg hatching of Argopecten irradians, and population growth of Brachionus plicatilis and Moina mongolica

The impacts of Prorocentrum donghaiense Lu and Alexandrium catenella Balech, causative species of the large-scale HAB in the East China Sea, were studied under laboratory conditions. According to bloom densities, the effects of monoculture and mixture of the two species were examined on the egg-hatching success of Argopecten irradians Lamarck, and the population growth of Brachionus plicatilis Muller and Moina mongolica Daday. The results showed that monoculture of A. catenella had a significant inhibition on the egg hatching success of A. irradians, and the population growth of B. plicatilis and M. mongolica. The median effective densities ( EDSo) inhibiting the egg hatching success of A. irradians for 24 h and the population growth of B. plicatilis and M. mongolica for 96 h were 800, 630, and 2 400 cells/cm(3), respectively. Monoculture of P. donghaiense has no such inhibitory effect on the egg hatching success of A. irradians; P. donghaiense at lower suitable densities could sustain the population growth of B. plicatilis (1 x 10(4) similar to 3 x 10(4)cells/cm(3)) and M. mongolica (2 x 10(4) similar to 5 x 10(4) cells/cm(3)); P. doaghaiense at higher densities had significantly adverse effect on the population growth of B. plicatilis (4 x 10(4) similar to 10 x 10(4) cells/cm(3)) and M. mongolica (10 x 10(4) cells/cm(3)). When the two algae were mixed according to bloom densities, P. donghaiense at suitable densities to some extent could decrease the toxicity of A. catenella to B. plicatilis and M. mongolica. The results indicated that the large-scale HAB in the East China Sea could have adverse effect on zooplankton, and might further influence the marine ecosystem, especially when there was also Alexandrium bloom.

Validation of the detection of Pseudo-nitzschia spp. using specific RNA probes tested in a microarray format: Calibration of signal based on variability of RNA content with environmental conditions.

Harmful algal blooms (HAB) occur worldwide and cause health problems and economic damage to fisheries and tourism. Monitoring for toxic algae is therefore essential but is based primarily on light microscopy, which is time consuming and can be limited by insufficient morphological characters such that more time is needed to examine critical features with electron microscopy. Monitoring with molecular tools is done in only a few places world-wide. EU FP7 MIDTAL (Microarray Detection of Toxic Algae) used SSU and LSU rRNA genes as targets on microarrays to identify toxic species. In order to comply with current monitoring requirements to report cell numbers as the relevant threshold measurement to trigger closure of fisheries, it was necessary to calibrate our microarray to convert the hybridisation signal obtained to cell numbers. Calibration curves for two species of Pseudo-nitzschia for use with the MIDTAL microarray are presented to obtain cell numbers following hybridisation. It complements work presented by Barra et al. (2012b. Environ. Sci. Pollut. Res. doi: 10.1007/s11356-012-1330-1v) for two other Pseudo-nitzschia spp., Dittami and Edvardsen (2012a. J. Phycol. 48, 1050) for Pseudochatonella, Blanco et al. (2013. Harmful Algae 24, 80) for Heterosigma, McCoy et al. (2013. FEMS. doi: 10.1111/1574-6941.12277) for Prymnesium spp., Karlodinium veneficum, and cf. Chatonella spp. and Taylor et al. (2014. Harmful Algae, in press) for Alexandrium.

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.

Phytoplankton in the Nhatrang Bay, South China Sea

Species composition, phytoplankton abundance, and relative yield of variable fluorescence (F_v/F_m) were determined in the mesotrophic Nhatrang Bay in October-November 2004. Species diversity (250 taxonomic units) and heterogeneity of the phytoplankton structure were high. With respect to number of species and their abundance, diatoms prevailed. In selected parts of the bay, dinoflagellates dominated. Average biomass in the water column under 1 m**2 (Bt) varied from 2.3 to 64.4 mg C/m**3 (av. 31.0 mg C/m**3). Bt values were the lowest at stations nearest to the river mouth. Seaward, Bt increased. Bt values increased with depth at some stations and decreased at others. In surface layers biomass was lower than that in the underlying waters. F_v/F_m values ranged from 0.10 to 0.64 (av. 0.49). The lowest F_v/F_m values were observed in the area close to the seaport. Over greater part of the bay, F_v/F_m values were higher than 0.47. Such values are indicative of relatively high potential of photosynthetic activity of phytoplankton. Abundance and species diversity were higher than those in the dry season (March-April).

Accumulation rates of dinoflagellate cysts at trap sites CB2, CB3, CB4, and CB9 off Cape Blanc

A 5-year sediment trap survey in the upwelling area off Cape Blanc (NW Africa) provides information on the seasonal and annual resting cyst production of dinoflagellates, their sinking characteristics and preservation potential. Strong annual variation in cyst production characterizes the region. Cyst production of generally all investigated species, including Alexandrium pseudogonyaulax (Biecheler) T. Horig. ex T. Kita et Fukuyo (cyst genus Impagidinium) and Gonyaulax spinifera (Clap. et J. Lachm.) Diesing (cyst genus Nematosphaeropsis) was enhanced with increasing upper water nutrient and trace-element concentrations. Cyst production of Lingulodinium polyedrum (F. Stein) J. D. Dodge was the highest at the transition between upwelling and upwelling-relaxation. Cyst production of Protoperidinium americanum (Gran et Braarud) Balech, Protoperidinium monospinum (Paulsen) K. A. F. Zonn. et B. Dale, and Protoperidinium stellatum (D. Wall) Balech, and heterotrophic dinoflagellates forming Brigantedinium spp. and Echinidinium aculeatum Zonn., increased most pronouncedly during upwelling episodes. Production of Protoperidinium conicum (Gran) Balech and Protoperidinium pentagonum (Gran) Balech cysts and total diatom valves were related, providing evidence of a predator-prey relationship. The export cyst-flux of E. aculeatum, P. americanum, P. monospinum, and P. stellatum was strongly linked to the flux of total diatom valves and CaCO3, whereas the export production of Echinidinium granulatum Zonn. and Protoperidinium subinerme (Paulsen) A. R. Loebl. correlated with total organic carbon, suggesting potential consumption of diatoms, prymnesiophytes, and organic matter, respectively. Sinking velocities were at least 274 m · d**-1, which is in range of the diatom- and coccolith-based phytoplankton aggregates and "slower" fecal pellets. Species-selective degradation did not occur in the water column, but on the ocean floor.