Responses of antioxidant system in Arabidopsis thaliana suspension cells to the toxicity of microcystin-RR

Microcystins are cyclic heptapeptide hepatoxins produced by many species of cyanobacteria. The toxic effects and mechanism of microcystins on animals have been well studied both in vivo and in vitro. It was also reported that microcystins had adverse effects on plants. However, to our knowledge, there is no information about the toxic effects and mechanism of microcystins on plant suspension cells. In this study, Arabidopsis thaliana suspension cells were exposed to a range dose of microcystin-RR. Lipid peroxidation, a main manifestation of oxidative damage, was studied and a time- and dose-dependent increase in malondiadehyde was observed. In contrast, glutathione (GSH) levels in the cells decreased after 48 h treatment with 1 and 5 mg/L of microcystin-RR. The activities of superoxide dismutase (SOD) and catalase (CAT) increased significantly after 48 h exposure to I and 5 mg/L of microcystin-RR, but glutathione S-transferase (GST) activity showed no difference compared with the control. These results clearly indicate that microcystin-RR is able to cause oxidative damage in A. thaliana suspension cells. Decrease of GSH content and increases of SOD and CAT activities reveal that the antioxidant system may play an important role in eliminating or alleviating the toxicity of microcystin-RR. The possible toxicity mechanism of microcystin-RR on the A. thaliana suspension cells is also discussed in this paper. (C) 2005 Elsevier Ltd. All rights reserved.

Selective control of toxic Microcystis water blooms using lysine and malonic acid: An enclosure experiment

Three enclosures (10 x 10 x 1.5-1.3 m in depth) were set beside Dianch Lake, Kunming, People's Republic of China, for the period from July 28 to August 26, 2002. The enclosures were filled with cyanobacterial (Microcystis aeruginosa) water bloom-containing lake water. Lake sediment that contained macrophytes and water chestnut seeds was spread over the entire bottom of each enclosure. Initially, 10 g/m(2) of lysine was sprayed in Enclosure B, and 10 g/m(2) each of lysine and malonic acid were sprayed together in Enclosure C. Enclosure A remained untreated and was used as a control. The concentrations of lysine, malonic acid, chlorophyll a, and microcystin as well as the cell numbers of phytoplankton such as cyanobacteria, diatom, and euglena were monitored. On day 1 of the treatment, formation of cyanobacterial blooms almost ceased in Enclosures B and C, although Microcystis cells in the control still formed blooms. On day 7 Microcystis cells in Enclosure B that had been treated with lysine started growing again, whereas growth was not observed in Microcystis cells in Enclosure C, which had been treated with lysine and malonic acid. On day 28 the surface of Enclosure B was covered with water chestnut (Trapa spp.) and the Microcystis blooms again increased. In contrast, growth of macrophytes (Myriophllum spicatum and Potamogeton crispus) was observed in Enclosure C; however, no cyanobacterial blooms were observed. Lysine and malonic acid had completely decomposed. The microcystin concentration on day 28 decreased to 25% of the initial value, and the pH shifted from the initial value of 9.2 to 7.8. We concluded that combined treatment with lysine and malonic acid selectively controlled toxic Microcystis water blooms and induced the growth of macrophytes. (c) 2005 Wiley Periodicals, Inc.

Responses of antioxidant systems in the hepatocytes of common carp (Cyprinus carpio L.) to the toxicity of microcystin-LR

The freshwater, bloom-forming cyanobacterium (blue-green alga) Microcystis aeruginosa produces a peptide hepatotoxin, which causes the damage of animal liver. Recently, toxic Microcystis blooms frequently occur in the eutrophic Dianchi Lake (300 km(2) and located in the South-Westem of China). Microcystin-LR from Microcystis in Dianchi was isolated and purified by high performance liquid chromatography (HPLC) and its toxicity to mouse and fish liver was studied (Li et al., 2001). In this study, six biochemical parameters (reactive oxygen species, glutathione, superoxide dismutase, catalase, glutathione peroxide and glutathione S-transferase) were determined in common carp hepatocytes when the cells were exposed to 10 mug microcystin-LR per litre. The results showed that reactive oxygen species (ROS) contents increased by more than one-time compared with the control after 6 h exposure to the toxin. In contrast, glutathione (GSH) levels in the hepatocytes exposed to microcystin-LR decreased by 47% compared with the control. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxide (GSH-Px) increased significantly after 6 h exposure to microcystin-LR, but glutathione S-transferase (GST) activity showed no difference from the control. These results suggested that the toxicity of microcystin-LR caused the increase of ROS contents and the depletion of GSH in hepatocytes exposed to the toxin and these changes led to oxidant shock in hepatocytes. Increases of SOD, CAT and GSH-Px activities revealed that these three kinds of antioxidant enzymes might play important roles in eliminating the excessive ROS. This paper also examined the possible toxicity mechanism of microcystin-LR on the fish hepatocytes and the results were similar to those with mouse hepatocytes. (C) 2003 Elsevier Science Ltd. All rights reserved.

The toxic effects of microcystin-LR on embryo-larval and juvenile development of loach, Misguruns mizolepis Gunthe

Microcystin-LR, a specific and potent hepatotoxin, was tested for its effects oil loach embryo-larval and juvenile development, The results of this study showed that loach embryos were more sensitive when exposed to microcystin-LR at a later than at an earlier stage of development, Juveniles were far less sensitive to MC-LR than were embryos and larvae. Mortality and developmental abnormality were proven to be dose-dependent and to be stage-specific sensitive. Among the abnormal changes noted were: pericardial edema and tubular heart, bradycardia, homeostasis, poor yolk resumption. small head, curved body and tail, and abnormal hatching, Liver and heart were the main targets of microcystin-LR toxicity. Ultrastructural analysis documented a complex set of sublethal effects of microcystin-LR on loach hepatocytes, chiefly including morphological alteration in nuclear and RER of loach liver cells. fit addition, microcystin-LR was lethal to loach juvenile in the subacute (7 days) exposure (LC50) = 593.3 mug/l). (C) 2002 Elsevier Science Ltd. All rights reserved.

Long-term toxic impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the reproduction, sexual differentiation, and development of different life stages of Gobiocypris rarus and Daphnia magna

The sexual ratio of Gobiocypris rarus exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 17 beta -estradiol from embryo to sexually mature revealed feminization and overdevelopment of connective tissue in male fish gonad in 2-30 pg/L TCDD concentration range. Daphnia magna was not sensitive to the high dose of TCDD (0.1-1000 ng/ml), but the reproduction of D. magna treated with TCDD decreased after the 8th day. 7-Ethoxyresorufin-O-deethylase (EROD) activities in newly fertilized eggs of G. rarus exposed to TCDD dosage groups (1000-100,000 pg/L) were significantly induced and increased with TCDD concentrations at the early life stage, while no difference was found between low TCDD dosage groups (<100 pg/L), but a good relationship between the EROD activity and the TCDD concentration was observed during a long-term developmental stage. There was a pericardial edema formed in a 2-week yolk-sac at the concentration of 1000 pg/L TCDD. But in the exposure group (2 pg/L TCDD for 120 days), the cell nuclei of hepatocytes was far from the center and packed toward the cell membrane; the cristae of most mitochondria in the cell dropped and collapsed; the rough endoplasmic reticulum broke into fragments; and numerous lipid droplets formed in the cell. (C) 2001 Academic Press.

瑞香狼毒（Stellera chamaejasme L.）对高寒草甸系统生长和非生长季节碳氮循环的影响及其花粉化感效应

瑞香狼毒（Stellera chamaejasme L.）是瑞香科（Thymelaeaceae）狼毒属的一种多年生野草，有毒。据调查，从20 世纪60 年代开始至今，狼毒在青藏高原东缘的高寒草甸上不断蔓延、密度不断变大，在一些地段甚至成为优势物种。有关狼毒在高寒草甸蔓延的生态系统效应的研究尚未见报道。本文从系统碳、氮循环的角度，分别研究狼毒在生长和非生长季节对高寒草甸生态系统的影响。同时，从花粉化感的角度，深入研究狼毒对当地同花期物种有性繁殖的影响。系统地研究高寒草甸生态系统物质循环过程，特别是非生长季节微生物和土壤碳氮库的动态变化，有助于揭示狼毒在系统物质循环方面的“物种效应”以及这种效应的季节变化，为丰富有关高海拔生态系统，特别是其非生长季的物质循环的科学理论做出贡献。同时，碳氮循环和花粉化感的研究还有助于深刻地理解狼毒作为一种入侵性很强的杂草的特殊的蔓延机制，从而为狼毒的有效防治、高寒草甸的科学管理提供依据。 针对狼毒在青藏高原高寒草甸上蔓延的生态系统碳氮循环方面的影响，开展以下2 方面的研究：（1）在生长季，研究松潘县尕米寺附近（北纬32°53'，东经103°40'，海拔3190 m）的两种地形（平地和阳坡）条件下狼毒对土壤碳氮循环影响及可能的原因。狼毒和其它几个主要物种（圆穗蓼（Polygonummacrophyllum D. Don var. Macrophyllum），草地早熟禾（Poa pretensis L.），四川嵩草（Kobresia setchwanensis Hand.-Maizz.），鹅绒委陵菜（Potentilla anserina L.var. anserine）和鳞叶龙胆（Gentiana squarrosa Ledeb.）的地上凋落物产量以及地上凋落物和根的化学组成被测量。在有-无狼毒斑块下，各种土壤的库（比如，铵态氮、硝态氮、无机磷和微生物生物量）和周转率（包括净矿化、净硝化、总硝化、反硝化和微生物呼吸速率）被测量和比较。（2）在非生长季节，尤其是春季冻融交替期，选取了两个研究地点——尕米寺和卡卡沟（北纬32°59'，东经103°41'，海拔3400 m），分别测定有狼毒和无狼毒斑块下土壤微生物生物量碳和氮、可溶性有机碳和氮以及铵态氮和硝态氮的动态变化。同时，分别在上述两个地点有-无狼毒的样地上，研究6 个主要物种（狼毒、圆穗蓼、草地早熟禾、四川嵩草、鹅绒委陵菜和鳞叶龙胆）从秋季开始、为期1 年的凋落物分解过程。 针对狼毒花粉化感对同花期其它物种可能的花粉化感作用开展以下工作：在实验室中，用一系列浓度的狼毒花粉水浸提液对与它同花期的其它物种以及自身花粉进行测试，测定花粉萌发率；在野外自然条件下的其它物种的柱头上施用上述浓度的狼毒花粉水浸提液，观测种子结实率，同时，观察狼毒花粉的种间花粉散布数量。 生长季节的研究结果表明，狼毒地上凋落物氮含量比其它几个主要物种更高，而木质素-总氮比更低。狼毒显著地增加其斑块下表层土壤中有机质的含量，而有-无狼毒的亚表层土壤在有机碳和总磷方面没有显著差异。狼毒表土中硝态氮含量在平地和阳坡比无狼毒土壤分别高113%和90%。狼毒表土中微生物生物量碳和氮量显著高于无狼毒表土。无论是平地还是阳坡，狼毒土壤的总硝化和微生物呼吸速率显著高于无狼毒土壤；而它们的反硝化速率只在平地有显著的差异。狼毒与其它物种间地上凋落物的产量和质量的差异可能是导致有-无狼毒土壤碳氮循环差异的原因。我们假设，狼毒可能通过增加贫氮生态系统土壤中的有效氮含量提高其入侵能力。 非生长季的研究结果表明，在青藏高原东缘的高寒草甸上，土壤微生物生3物量在11 月的秋-冬过渡期达到第一个峰值；在春季的冻融交替期，微生物生物量达到第二个峰值后又迅速降低。无机氮以及可溶性有机碳氮与微生物生物量有相似的变化过程。微生物碳氮比呈现显著的季节性变化。隆冬季节的微生物生物量碳氮比显著高于生长旺季的微生物碳氮比。这种变化可能暗示冬、夏季微生物的群落组成和对资源的利用有所不同。有-无狼毒斑块下土壤微生物和土壤碳、氮库一般只在秋-冬过渡期有显著差异，有狼毒土壤微生物生物量和土壤碳、氮库显著高于无狼毒土壤；而在之后的冬季和春季没有显著差异。所有6 个物种凋落物在非生长季分解率为24%-50%，均高于生长季的10%到30%。其中在秋-冬过渡期，凋落物开始埋藏的两周时间内，分解最快，达10%-20%。不同物种凋落物全年的分解率和分解过程有显著差异。圆穗蓼在全年的分解都较缓慢（非生长季26%，生长季15%），草地早熟禾和四川嵩草等全年的分解速率比较均匀（非生长季和生长季均为30%，非生长季略高），而狼毒在非生长季分解较快（约50%），而在接下来的生长季分解变得缓慢（约12%）。所有物种的凋落物氮含量在非生长季下降，而在随后的生长季上升。 实验室的花粉萌发试验证明，狼毒花粉对自身花粉萌发没有自毒作用，而其它受试的所有物种（圆穗蓼，秦艽（Gentiana macrophylla Pall. var. fetissowii），湿生扁蕾（Gentianopsis paludosa (Hook. f.) Ma var. paludosa），鳞叶龙胆，椭圆叶花锚（Halenia elliptica D. Don var. elliptica），蓝钟花（Cyananthus hookeri C. B.Cl. var. grandiflorus Marq.），小米草（Euphrasia pectinata Ten.），川西翠雀花（Delphinium tongolense Franch.），高原毛茛（Ranunculus tanguticus (Maxim.)Ovcz. var. tanguticus）和鹅绒委陵菜）的花粉萌发率随着狼毒花粉浸提液浓度的增加呈显著的非线性降低。大约3 个狼毒花粉的浸提液就可以抑制受试的多数物种的50%的花粉萌发。在鳞叶龙胆和小米草柱头上狼毒花粉的数量分别为5.76 个和3.35 个。狼毒花粉散布数量的差异最可能的原因在于是否有共同的传粉昆虫。花的形状（辐射对称VS 左右对称）、植株或花的密度以及花期重叠性可以部分解释这种差异。在野外试验中，我们发现6 个物种（秦艽、湿生扁蕾、鳞叶龙胆、椭圆叶花锚、蓝钟花和小米草）的种子结实率随狼毒花粉浸提液浓度的增加呈显著的非线性降低。鳞叶龙胆和小米草柱头上狼毒花粉的数量（分别是5.76 个和3.35 个）分别达到了抑制它们63%和55%种子结实率的水平。因此，狼毒对鳞叶龙胆和小米草可能存在明显的花粉化感抑制作用。狼毒周围的物种可能通过花期在季节或昼夜上的分异避免受到狼毒花粉化感的影响或者通过无性繁殖来维持种群繁衍，因此狼毒通过花粉化感作用对其周围物种繁殖的影响程度还需要进一步地研究。如果狼毒的花粉化感抑制作用确实存在，那么它可能成为一种自然选择压力，进而影响物种的进化。 Stellera chamaejasme L., a perennial toxic weed, has emerged and quicklydominated and spread in the high-frigid meadow on the eastern Tibetan Plateau ofChina since the 1960s. In the present study, effects of S. chamaejasme on carbon andnitrogen cycles on the high-frigid meadow on the eastern Qinghai-Tibetan Plateau ingrowing and non-growing season, and its pollen allelopathic effects on the sympatricspecies were determined. The present study that focused on carbon and nitrogencycles, especially on microbial biomass and pools of carbon and nitrogen innon-growing season, could profoundly illuminate plant-species effects on carbon andnutrient cycles and its seasonal pattern and help to understand spread mechanism ofS. chamaejasme as an aggressive weed. The present study also contributed to furtherunderstand carbon and nutrient cycles on alpine regions in non-growing season andprovide a basis on weed control of S. chamaejasme and scientific management in thehigh-frigid ecosystem. Effects of S. chamaejasme on carbon and nitrogen cycles on the high-frigidmeadow on the eastern Qinghai-Tibetan Plateau were determined. The study couldbe divided into two parts. (1) In the growing season, we quantified the effects of S.chamaejasme on carbon and nitrogen cycles in two types of topographic habitats, theflat valley and the south-facing slope, where S. chamaejasme was favored to spreadlitter and root were measured to explain the likely effects of S. chamaejasme on soilcarbon and nutrient cycles. The sizes of various soil pools, e.g. nitrite, ammonium,inorganic phosphorus and microbial biomass, and turnover rates including netmineralization, gross nitrification, denitrification and microbial respiration weredetermined. (2) In the non-growing season study, microbial biomass carbon andnitrogen, soluble organic carbon and nitrogen, ammonium and nitrate weredetermined through the non-growing season, especially in the processes offreeze-thaw of spring in two high-frigid sites, i.e. Kaka valley and Gami temple, onthe eastern Qinghai-Tibetan Plateau. Meanwhile, litter decomposition of six commonspecies, including Stellera chamaejasme L., Polygonum macrophyllum D. Don var.Macrophyllum, Poa pretensis L., Kobresia setchwanensis L., Potentilla anserina L.var. anserine and Gentiana squarrosa Ledeb., were also examined under theabove-mentioned experimental design through one whole-year, which began in theautumn in 2006. In the study of pollen allelopathy, several work, including in vitro study oneffects of extract of pollen from S. chamaejasme on sympatric species and pollenfrom itself, field experiments on effects of pollen extract with the same regime ofconcentrations on seed set and field observation on heterospecific pollen transfer ofS. chamaejasme to six of those sympatric species has been done. The results in the growing season showed that aboveground litter of S.chamaejasme had higher tissue nitrogen and lower lignin: nitrogen ratio than thoseco-occurring species. S. chamaejasme significantly increased topsoil organic matter,whereas no significant differences were found for organic C and total P in subsoilbetween under-Stellera and away-Stellera locations. The nitrate in Stellera topsoilwas 113% and 90% higher on the flat valley and on the south-facing slope,respectively. Both microbial biomass C and N were significantly higher in Stelleratopsoil. Gross nitrification and microbial respiration were significantly higher inStellera topsoil both on the flat valley and on the south-facing slope, whereassignificant differences of denitrification were found only on the flat valley. Thedifferences in the quantity and quality of aboveground litter are a likely mechanismresponsible for the changes of soil variables. We assumed that S. chamaejasme couldenhance their spread by increasing nutrient availability in N-deficient ecosystems. The results in the non-growing season showed that microbial biomass achievedthe first summit in late autumn and early winter on the eastern Qinghai-TibetanPlateau. In the stages of freeze-thaw of spring, microbial biomass firstly achieved thesecond summit and subsequently sharply decreased. Inorganic nitrogen, solubleorganic carbon and nitrogen had a similar dynamics with that of microbial biomass.Ratio of microbial biomass carbon and nitrogen had an obviously seasonal pattern.The highest microbial C: N were in the non-growing season, which weresignificantly higher than those in the growing season. The seasonal pattern inmicrobial biomass C: N suggested that large changes in composition of microbialpopulation and in resources those used by microbes between summer and winter.Generally, microbial biomass and pools size of carbon and nitrogen in Stellera soilwere significantly higher than those under adjacent locations in late autumn andearly winter, but there were not significant differences in winter and in spring. Litterof all the focal species (Stellera chamaejasme L., Polygonum macrophyllum D. Donvar. Macrophyllum, Poa pretensis L., Kobresia setchwanensis Hand.-Maizz.,Potentilla anserina L. var. anserine and G. squarrosa Ledeb.) decomposed about24%-50% in the non-growing season, which were higher than those in the growingseason (ranged from 10% to 30%). Litter decomposed 10%-20% within the first twoweeks in late autumn and early winter. Significant differences in the whole-yeardecomposition rate and in the processes of decomposition were found among species.Polygonum macrophyllum decomposed slowly through the whole year (26% and15% in the non-growing season and in the growing season, respectively). Certainspecies, such as P. pretensis L. and K. setchwanensis, decomposed at a similar rate(30% both in the non-growing and in the growing season, slightly higher in the8growing season than those in the growing season), whereas S. chamaejasmedecomposed more rapidly (about 50%) in the non-growing season and subsequentlydecomposition became slow (about 12%) in the growing season. Litter nitrogencontents of all the focal species firstly decreased in the non-growing season and thenincreased in the growing season. In vitro experiments of pollen allelopathy, the results showed that pollen from S.chamaejasme was not autotoxic, whereas pollen germination in all the sympatricspecies (Polygonum macrophyllum D. Don var. Macrophyllum, Gentianamacrophylla Pall. var. fetissowii, Gentianopsis paludosa (Hook. f.) Ma var. paludosa,Gentiana squarrosa Ledeb., Halenia elliptica D. Don var. elliptica, Cyananthushookeri C. B. Cl. var. grandiflorus Marq., Euphrasia pectinata Ten., Delphiniumtongolense Franch., Ranunculus tanguticus (Maxim.) Ovcz. var. tanguticus andPotentilla anserina L. var. anserina) decreased nonlinearly as the increasingconcentrations of extract of pollen from S. chamaejasme. Pollen Extract of threepollens from S. chamaejasme generally inhibited 50% pollen germination of most ofthe focal species. 5.76 and 3.35 pollens from S. chamaejasme were observed in fieldon stigmas of G. squarrosa and E. pectinata, respectively. Differences inheterospecific pollen transfer of S. chamaejasme could be attributed to the primaryreason whether they shared common pollinators. Flower morphology (e.g.zygomorphic or actinomorphic), plant or floral density and concurrence in floweringphonologies could explain, in part, the differences in heterospecific pollen transfer.In field experiments, the results showed that seed set in six sympatric species(Gentiana macrophylla Pall. var. fetissowii, Gentianopsis paludosa (Hook. f.) Mavar. paludosa, Gentiana squarrosa Ledeb., Halenia elliptica D. Don var. elliptica,Cyananthus hookeri C. B. Cl. var. grandiflorus Marq. and Euphrasia pectinata Ten.)decreased nonlinearly as the increasing concentrations of extract of pollen from S.chamaejasme. According to the nonlinear curves, the amounts of pollens from S.chamaejasme on stigmas of G. squarrosa and of E. pectinata (i.e. 5.76 grains and3.35 grains, respectively) could reduce 63% and 55% seed set of G. squarrosa and ofE. pectinata, respectively. Thus, allelopathic effects of S. chamaejasme on G.squarrosa and E. pectinata could be realistic. The sympatric species of S.chamaejasme could avoid pollen allelopathy of S. chamaejasme to sustainthemselves. This highlights the need to study how much pollen allelopathy in S.chamaejasme influences the sympatric species through divergence in seasonal ordiurnal flowering phonologies or through asexual reproduction. If pollen allelopathyin S. chamaejasme was confirmed, it could be as a pressure of natural selection andthus play an important role in species evolution.

溶藻菌群对铜绿微囊藻的溶藻效应及其作用机理研究

水华暴发是一个世界性的问题，近年来在发展中国家显得尤其严重。水华暴发给环境和公众健康带来巨大灾难，一些蓝藻产生的毒素可以造成鱼类、鸟禽和家畜的死亡，而臭名昭著的微囊藻产生的微囊藻毒素更是有强烈致癌效应。因此，寻找控制水华藻类的有效方法非常迫切。在利用物理和化学方法处理不甚理想的情况下，利用溶藻细菌控藻成为一个新的研究方向。溶藻细菌一般直接从富营养化水体中分离，杀藻活力对有害蓝藻具有较强的选择性而不危害其它生物，尤其适合在水华发生初期使用，可以在短时间内达到阻止藻类增殖的效果。本研究富集分离到一个高效溶解铜绿微囊藻的溶藻菌群，对其溶藻效应和溶藻机制进行了探索研究。 1溶藻菌群的富集筛选及其溶微囊藻效果 富集筛选得到一个有明显抑藻效果的菌群，它对铜绿微囊藻有显著溶藻效果。与对照组相比，加入富集的溶藻菌后，第4 d开始出现溶藻现象，6~8 d出现明显的溶藻效果，8 d后测得叶绿素去除率在85%以上。 2 溶藻菌群的作用范围及溶藻特性 富集分离到的溶藻菌群对铜绿微囊藻和念珠藻有显著溶藻作用，对水华微囊藻和其它几株受试微囊藻没有明显溶藻效应。该溶藻菌群不仅可以在液体中溶解铜绿微囊藻，生长在固体平板上的藻苔也有一定的溶藻效应，生成溶藻空斑。保证快速溶藻的最大稀释度可以达到1/100, 000。 3 环境因子对菌群溶藻效力的影响 试验发现，不同的pH、温度、和光照条件下，溶藻菌群溶藻效力明显不同，且不同种类的氮源对其溶藻作用也有一定影响。这些条件对该菌群溶藻作用的影响，在相当的程度上可能取决于它们对藻和细菌两者的生长状况的影响综合。 4 溶藻菌群的溶藻作用机理 溶藻菌液过滤除菌和煮沸灭菌处理后溶藻液，未见明显的溶藻效果，只有原液具有很好的溶藻效果。因此可初步确定，蓝藻细胞的溶解可能是由溶藻菌直接接触藻细胞产生的作用效果。显微镜观察发现，细菌在溶藻的过程中频繁地接触藻细胞并侵入藻细胞，破坏进而裂解杀死藻细胞。这也进一步说明了此溶藻菌是通过直接方式杀藻。 5 溶藻菌群的菌群结构解析 分离有溶藻效果的纯菌的多次尝试都没有成功。结合DGGE和16S rDNA文库综合分析发现：Rubritepida菌，假单胞菌和鞘氨醇单胞菌是存在于铜绿微囊藻中的三种伴生细菌。加入富集的溶藻菌群后，菌群结构发生明显的变化，Rubritepida菌、假单胞菌消失，混合菌群则包含未培养黄杆菌，鞘氨醇单胞菌和噬氢菌，其中黄杆菌是优势菌群，并且细菌种群结构的变化与藻细胞消亡之间有显著的相关性。通过菌种的分离鉴定与DGGE和16S rDNA文库的测序结果比较，一些未培养菌可能在溶藻过程中起重要调控作用。 6 溶藻细菌控藻应用基础 (1) 扩大规模的模拟水华实验进一步确定了细菌对微囊藻的强烈溶解作用。 (2) 铜绿微囊藻(Microcystis aeruginosa 905, zc)、微囊藻(Microcystis spp., zd)和溶藻菌群共培养试验表明，zc可以抑制zd生长，而溶藻菌群可以溶zc。 本研究是第一次报道混合菌群的溶藻效应。该溶藻菌群对带有藻际细菌的铜绿微囊藻具有高效的溶藻效力，表明它对自然界中存在的带菌铜绿微囊藻和其它一些蓝藻的生消具有一定的控制作用。对进一步研究菌藻关系与生态学作用，以及对富营养化湖泊和水库水体中蓝藻暴发的防控，该菌群具有一定的应用潜力。 Cyanobacterial blooms break out frequently all over the world, especially in developing countries. Blooms create enormous disasters to public health and to the environment. Some cyanobacterial blooms produce extremely toxic substances that have killed fish, domestic animals and birds. It has been well known that microcystins, a hepatoxin produced by Microcystis, can promote tumors in humans. So it is very important to find an effective method for controlling the growth of the bloom-forming algae. Measures for controlling such kind of algae include physical, chemic and biologic means, but the former two may damage the aquatic environment and require high-energy inputs. The alternative approach for the elimination of nuisance algae involves the application of algicidal bacteria. The algicidal bacteria, which are nontoxic to other organisms and most of which are isolated from the eutrophic lake in situ, may be potential microbial algaecides. In the initial stages of the water blooms, they are able to restrain the biomass or multiplication of the bloom-forming algae in a short time. In order to use algicidal bacteria to suppress blooms of M. aeruginosa, we isolated a bacterial culture capable of lysing the noxious cyanobacteria M. aeruginosa. In this paper we described some properties of the bacterial culture and its growth-inhibiting or algicidal effects on the growth of M. aeruginosa, and investigated its algicidal mechanisms. 1 Enrichment of a microbial culture that lyses Microcystis aeruginosa A mixed bacterial culture was isolated from a hypereutrophic pond and showed significant algicidal activity against the noxious Microcystis aeruginosa. Algae lysis would be seen obviously 4 days later when the algae culture was killed and became yellow contrast to no-addition controls, and chlorophyll a (chl-a) reduction went beyond 85% 8 days later. 2 The host range and some other algicidal feature of the mixed algicidal culture. Microcystis aeruginosa, Nostoc sp., were susceptible to the mixed algicidal culture, while the lytic effects of this mixed culture on Microcystis flos-aquae and some other tested Microcystis were feeble.The algicidal culture can not only lyse M. aeruginosa in liquid media, but aslo lyse M. aeruginosa lawns on soft agar plates and form plaques. The maximun dilution of the mixed culture required for rapid Microcystis lysis is 1/100, 000. 3 Influences of environmental factors such as pH, temperature, illumination, and the nitrogen source on the lytic activity of the mixed bacterial culture on Microcystis aeruginosa. In our investigations, it was shown that the lytic activity of the mixed bacterial culture on Microcystis aeruginosa was straightly correlated with pH, temperature, illumination, as well as the nitrogen source in the medium. The impacts of these environmental factors on the algicidal activity of the mixed bacterial culture, to a certain extent, may depend on both the algal and the bacterial growth rates under the tested environmental conditions. 4 The mechanisms of algal cell lysis by the algicidal bacteria Death was detected when the mixed bacterial culture was added to the algal culture, but not when only the culture filtrate or autoclaved bacterial culture was added. This indicates that the mixed bacterial culture did not release extracellular products inhibitory to Microcystis aeruginosa. In addition, under the microscope, we observed frequent contacts btween bacteria and algae cells, and some bacteria can even penetrate into target algal cells and destroyed them. These results may suggest that the bacterium kill the alga by direct contact. 5 Molecular Characterization of the algicidal bacterial culture Attempts for isolation of pure bacterium or bacteria from the enrichment culture responsible for Microcystis lysis have so far been failed. Based on PCR-DGGE (denaturing gradient gel electrophoresis) and 16S rDNA clone library analysis, Rubritepida sp., Pseudomonas sp. and Sphingomonas sp., as accompanying bacteria, were existed in M. aeruginosa. The bacterial community in M. aeruginosa showed significant change after adding the enrichment culture, where uncultured Flavorbacterium sp., Sphingomonas sp. and Hydrogenophaga sp. were observed, and the uncultured Flavorbacterium sp. became a dominant species. The obvious correlation can be seen between change of bacterial population and extinction of M. aeruginosa. Compared identification of pure bacterium with sequencing of DGGE bands and the clone distribution of the clone libraries, it was inferred that some uncultured bacteria were probably play an important role in controlling the growth and abundance of M. aeruginosa. This report is the first example of a mixed bacterial culture with the ability to lyse M. aeruginosa. 6 Further study for algae control by applications of algicidal bacteria (1) Algae lysis would be seen obviously 6 days later when the algae culture was killed and became yellow contrast to no-addition controls, and chlorophyll a (chl-a) was reducted to a low level 20 days later in the simulated water bloom experiments. (2) The growth of Microcystis sp. (zd) was restrained by Microcystis aeruginosa 905 (zc) when they were co-cultured together, and zc was lysed by the algicidal bacterial culture. This report is the first example of a mixed bacterial culture with the ability to lyse M. aeruginosa, and its algicidal activity remained high against non-axenic tested M. aeruginosa, suggesting that bacteria in the natural environment could play a role in controlling the growth and abundance of M. aeruginosa and other cyanobacteria. Such bacteria could also potentially be used as agents to prevent the mass development of cyanobacteria in eutrophic lakes and reservoirs.

Direct toxicity assessment of toxic chemicals with electrochemical method

Electrochemical measurement of respiratory chain activity is a rapid and reliable screening for the toxicity on microorganisms. Here, we investigated in-vitro effects of toxin on Escherichia coli (E. coli) that was taken as a model microorganism incubated with ferricyanide. The current signal of ferrocyanide effectively amplified by ultramicroelectrode array (UMEA), which was proven to be directly related to the toxicity. Accordingly, a direct toxicity assessment (DTA) based on chronoamperometry was proposed to detect the effect of toxic chemicals on microorganisms. The electrochemical responses to 3,5-dichlorophenol (DCP) under the incubation times revealed that the toxicity reached a stable level at 60 min, and its 50% inhibiting concentration (IC50) was estimated to be 8.0 mg L-1. At 60 min incubation, the IC50 values for KCN and As2O3 in water samples were 4.9 mg L-1 and 18.3 mg L-1, respectively. But the heavy metal ions, such as Cu2+ Pb2+ and Ni2+, showed no obvious toxicity on E. coli.

Three tetraspanins from Chinese shrimp, Fenneropenaeus chinensis, may play important roles in WSSV infection

Three members of the tetraspanin/TM4SF superfamily were cloned from Chinese shrimp, Fenneropenaeus chinensis. The deduced amino acid sequences of the three proteins have typical motifs of the tetraspanin/TM4SF superfamily. Phylogenetic analysis of the proteins, together with the known tetraspanins of invertebrates and vertebrates, revealed that they belong to different tetraspanin subfamilies: CD9, CD63 and tetraspanin-3. The three cloned genes of CD9, CD63 and tetraspanin-3 showed apparently different tissue distributions. The CD9 gene (FcCD9) was specifically expressed in the hepatopancreas. While for the CD63 gene (FcCD63), the highest expression was detected in nerves, epidermis and heart, with low expression in haemocytes, ovary, gill, hepatopancreas and stomach and no expression in intestine, muscle and lymphoid organ. Compared with FcCD9 and FcCD63, the tetraspanin-3 gene (FcTetraspanin-3) was more broadly expressed and its highest expression was detected in the intestine. Its expression in nerves was lower than in the intestine, but was higher than in other tissues. Expression in haemocytes, ovary and muscle was much lower than in other tissues. The expression profiles of FcCD9, FcCD63 and FcTetraspanin-3 in different tissues, including haemocytes, lymphoid organ and hepatopancreas, were compared by real-time PCR when shrimp were challenged by live white spot syndrome virus (WSSV) and heat-inactivated WSSV. All three tetraspanins were markedly up-regulated in the live WSSV-challenged shrimp tissues. The data suggested that the three cloned members of TM4SF superfamily in Chinese shrimp may play a key role in the route of WSSV infection.