米氏凯伦藻在三种无机氮源的生长情况

米氏凯伦藻引发的赤潮危害鱼类和无脊椎动物,给养殖业带来严重的经济损失,研究其营养生理生态,对掌握该藻形成赤潮的规律有着重要的意义。采用一次培养的实验方法研究了米氏凯伦藻(Karenia mikimotoi Hansen)在三种无机氮源不同氮磷浓度比(cN/cP)下的生长情况。结果表明:在分别以氯化铵、硝酸钠和亚硝酸钠为氮源时,米氏凯伦藻生长的最适cN/cP比分别为32、32和100。米氏凯伦藻对氯化铵的利用效率相对低于硝酸钠和亚硝酸钠,以氯化铵为氮源该藻生长缓慢,细胞密度低;而分别以硝酸钠和亚硝酸钠为氮源时该藻生长情况较好,可较长时间维持高的细胞密度。     

不同氮源对4种海洋微藻生长的影响

采用实验室一次性培养的方法,研究了硝氮、氨氮、尿素和混合氨基酸等4种不同氮源对典型赤潮藻赤潮异弯藻Heterosigma akashiwo、凯伦藻Karenia sp.、球形棕囊藻Phaeocystis globosa和常见浮游植物优势种类角毛藻Chaetoceros sp.生长的影响。结果表明,这4种海洋微藻不仅能利用无机氮硝氮和氨氮,而且也均能利用有机氮尿素和混合氨基酸。赤潮异弯藻、凯伦藻和角毛藻均在以硝氮为唯一氮源时,比生长速率分别达到最大值0.45、0.52和0.70 d-1;而球形棕囊藻在以硝氮和尿素为唯一氮源时,比生长速率均达最大值0.65 d-1。可溶性有机氮库中的重要组成成分尿素和氨基酸均能显著促进4种海洋微藻的生长;相比较而言,赤潮异弯藻和凯伦藻更加喜好有机氮氨基酸,而球形棕囊藻和角毛藻更加喜好尿素。海洋微藻具备利用有机氮源的能力,无疑扩展了其氮营养来源,在无机氮缺乏而有机氮丰富的水体中,它们在浮游植物群落中更具有竞争优势。     

米氏凯伦藻对蒙古裸腹溞的毒性及致毒途径分析

米氏凯伦藻(Karenia mikimotoi Hasen)是一种典型的鱼毒性赤潮藻,近年来在我国频繁爆发,给水产养殖业带了严重的经济损失.在实验室条件下,采用混合培养、隔离培养等手段研究了米氏凯伦藻及不同细胞组分对枝角类蒙古裸腹溞(Moina mongolica)摄食与生存的影响,分析了其致毒途径.结果表明,随着混合...     

珠江口海域3种重要有害藻类的溶血毒性研究

从珠江口海域分离、鉴定出3种重要有害藻类小普林藻JX12(Prymnesium parvum)、剧毒卡尔藻JX24(Karlodinium veneficum)、红色赤潮藻JX14(Akashiwo sanguinea),在实验室条件下研究了不同反应温度和pH值对小普林藻溶血活性的影响,在此基础上对海洋微藻溶血活性的测定方法进行了优化,并进一步分析比较了不同藻株以及不同生长时期溶血毒性的变化特征。研究结果显示,在实验温度范围内(4~50°C),小普林藻的溶血活性随温度的升高而增大,37℃为其最佳反应温度,pH 8和50 min为其最佳反应条件。不同生长时期的小普林藻溶血毒性具有显著差异,对数期溶血活性(5.67×10~(-7)HU·cell~(-1))显著高于稳定期(2.32×10~(-7)HU·cell~(-1))和衰亡期(3.40×10~(-7)HU·cell~(-1))。分离自珠江口海域的3种微藻均检测出溶血毒性,单个细胞溶血活性由强到弱分别为红色赤潮藻(976.20×10~(-7)HU·cell~(-1))、小普林藻(5.67×10~(-7)HU·cell~(-1))、剧毒卡尔藻(2.58×10~(-7)HU·cell~(-1))。值得注意的是,红色赤潮藻中国株JX14的单位细胞溶血活性显著高于美国株AS2,是后者的2倍以上。本研究首次确认珠江口海域红色赤潮藻、小普林藻和剧毒卡尔藻均具有较强的溶血毒性,这些有害藻类一旦形成赤潮可能对河口生态系统安全以及水产养殖业造成严重危害。     

4种典型有害赤潮原因种的溶血特性研究

对4种典型赤潮原因种的细胞和培养水中的溶血特性进行了研究.藻种分别是:赤潮异弯藻(Heterosigma akashiwo)的两个品系,微小亚历山大藻(Alexandrium minutum),东海原甲藻(Prorocentrum donghaiense)和微小卡罗藻(Karlodinum micrum).对各藻类的去藻上清液和超声破碎细胞提取液进行了溶血性检测,发现:几种赤潮种对鲫鱼红血细胞的溶血特性具有种间差异,同藻种的细胞与其培养水中的溶血强度存在差异.具体表现为:赤潮异弯藻具有最强的溶血性,去藻上清液的溶血性(60￣100%)远大于超声破碎细胞提取液的溶血性(30￣60%);微小卡罗藻超声破碎细胞提取液的溶血性(70%)大于去藻上清液的溶血性(30%),且是各藻超声破碎细胞提取液溶血性中最强的;东海原甲藻和微小亚历山大藻溶血性较弱,微小亚历山大藻去藻上清液的溶血性(30%)略大于超声破碎细胞提取液的溶血性(10%),东海原甲藻两部分的溶血性差异不显著.赤潮种类及其细胞内外的溶血特性在一定程度上显示了赤潮原因种在赤潮致害过程中的特征.     

珠江口双胞旋沟藻Cochlodinium geminatum赤潮生消过程的环境特征初步分析

为了解2009年珠江口双胞旋沟藻赤潮生消过程,探索其生消机制,对赤潮进行了连续10 d(10月28日─11月6日)的跟踪监测,设置了3条监测断面,共12个站位。通过现场测定和实验室分析,研究赤潮藻密度、叶绿素a、营养盐、温度、盐度、p H和溶解氧的变化规律。赤潮区双胞旋沟藻的密度先升高后降低,最高密度达到2.765×107 cells·L-1,叶绿素a含量呈先升后降的趋势,最大值达到172.76μ·L-1,监测海域无机氮和无机磷均处于富营养化水平,无机氮表现出先增加后减少,在赤潮末期稍有回升的趋势,无机磷总体呈现下降趋势。赤潮区水温变化范围为19.33~27.66℃,盐度的变化范围为9.16~31.5,p H的变化范围为7.45~8.51,溶解氧的变化范围为5.50~11.45 mg·L-1。通过对赤潮期间的环境因子分析,并结合2006年该海域发生的双胞旋沟藻赤潮事件和近几年周边海域发生赤潮的情况,得出以下结论:2009年珠海双胞旋沟藻赤潮规模较大,是中国沿海由双胞旋沟藻引起的最大规模赤潮。10月25日─10月27日为赤潮的起始发展阶段,10月28日─11月1日为赤潮形成维持阶段,11月2日─11月6日为赤潮消亡阶段。赤潮生消过程浮游植物群落结构发生了变化,赤潮持续期双胞旋沟藻占主要优势,赤潮消亡期间硅藻(骨条藻Skeletonema spp.和角毛藻Chaetoceros spp.)数量增加,演替双胞旋沟藻成为优势种。此次赤潮事件中有机氮浓度较高,约占总氮的60%,是赤潮藻大量繁殖代谢所致。研究海域处于严重富营养化水平,这是诱发赤潮发生的基础。同时,长时间处于高温、低盐的环境,使双胞旋沟藻急剧繁殖,诱发赤潮。在赤潮后期,恶劣的气象条件是赤潮消退的主要原因。     

BDE-47对4种海洋微藻抗氧化酶活性的影响

运用实验生态学和生物化学的方法,研究了不同质量浓度的2,2',4,4'-四溴联苯醚(BDE-47)对海水小球藻Chlorellaautotropica,牟氏角毛藻Chaetoceros muelleri,中肋骨条藻Skeletonema costatum和赤潮异弯藻Heterosigam akashiwo的抗氧化酶活性的影响.结果表明:在实验设定质量浓度范围内(0.1～2.5 μg·L~(-1)),4种微藻的超氧化物歧化酶(SOD)和过氧化氢酶(CAT)均能出现一定的应激活性,以减少BDE-47胁迫对藻细胞自身的危害,但是酶活增加的幅度却有很大不同.与赤潮异弯藻和中肋骨条藻相比,海水小球藻和牟氏角毛藻的SOD和CAT对BDE-47具有较高的敏感性.4种海洋微藻的SOD和CAT对BDE-47的敏感性顺序依次为:海水小球藻>牟氏角毛藻>中肋骨条藻>赤潮异弯藻.因此,海水小球藻的SOD和CAT可以作为指示海洋环境中BDE-47污染水平的生物标志物.     

除菌处理对强壮前沟藻和青岛大扁藻空长的影响

对微藻除菌以及未除菌与除菌后微藻生长特性的变化进行了研究.采用抗生素(新霉素、链霉素和卡纳霉素)组合加入的方法,对微藻(强壮前沟藻和青岛大扁藻)进行了无菌化处理.通过吖啶橙染色,镜检观察到与藻细胞共生的细菌经抗生素处理后数最明显减少,从而成功获得了2种无菌海洋微藻的纯藻种.比较了抗生素除菌前后环境细菌对微藻生长的影响,结果表明,除菌后的强壮前沟藻与未除菌的相比,推迟2 d进入稳定期,处于稳定期的最大细胞密度比未除菌的提高了24%.除菌后的强壮前沟藻和青岛大扁藻不易老化,可保持良好的悬浮状态.图7表1参16     

混合培养对城市污水厂二级出水培养能源微藻的生长促进作用

利用城市污水培养能源微藻可以实现水质净化和生物质生产的耦合,备受关注。生物质生产效率较低是限制其大规模应用的主要因素之一,混合培养是提高微藻生物质产率的一种潜在方法。为筛选出城市二级出水条件下合适的能源微藻混合藻种,考察了二级出水条件下3株高含油脂藻种栅藻LX1（Scenedesmussp. LX1）、椭圆小球藻YJ1（Chlorella ellipsoidea YJ1）和雨生红球藻（Haematococcus pluvislis）单一藻种和两两混合培养时的生长特性,比较了各微藻单一藻种及两两混合培养时的生长特性参数及生物质产量。研究结果表明,3种微藻的两两混合组合均能在二级出水条件下正常生长,各微藻干物质量浓度在第10天左右均能达到100 mg·L^-1左右。与栅藻LX1和椭圆小球藻YJ1相比,雨生红球藻在两两混合培养条件下表现出更高的藻细胞干物质量浓度增长趋势。3种微藻的内禀生长速率均显著高于各自的单一培养,栅藻 LX1和雨生红球藻混合培养时分别达到最高内禀增长速率（分别为1.36 d^-1、0.97 d^-1）。栅藻LX1与雨生红球藻混合培养时比生长速率分别为0.59 d^-1和0.42 d^-1,分别比栅藻LX1和雨生红球藻的单一藻种培养提高了36%、9.0%。与单一藻种相比,混合培养促进了微藻的生物质产量,栅藻LX1和雨生红球藻混合藻种的生物质产量（277 mg·L^-1）分别比栅藻LX1、雨生红球藻的单一藻种培养提高了64%和42%。栅藻LX1与雨生红球藻藻种组合具备作为二级出水条件下能源微藻培养合适混合藻种的潜力。     

荧光法监测硝普钠释放NO过程及其对微藻生长的影响（Fluorescence Dynamic Monitoring of Nitric Oxide Release Process Following Administration of Sodium Nitroprusside and Its Effect on the Growth of Microalga）

以硝普钠(Sodium nitroprusside,SNP)为一氧化氮(Nitric oxide,NO)供体研究了NO对海洋微藻生长的影响.对不同浓度SNP在海水介质中释放NO的过程进行了监测;对所培养的亚心形扁藻(Platymonas subcordiformis)和中肋骨条藻(Skeletonema costatum)进行藻密度测定,观测NO对微藻生长的影响.结果表明:5、10和100μmol·L-1的SNP释放NO浓度大约分别为6×10-9、9×10-9和2×10-7mol·L-1左右,而释放时间分别为4、5.5和7.5h.研究表明,NO对不同微藻有明显不同的作用规律:NO持续作用下,对亚心形扁藻的最佳作用浓度在10-8mol·L-1数量级;对赤潮藻中肋骨条藻的最佳作用浓度在10-9mol·L-1数量级;赤潮藻对NO的响应比非赤潮藻更灵敏,NO可能是海洋生态系中微藻生长重要的调节因子.     

环介导等温扩增技术(LAMP)检测人星状病毒方法的建立及其在再生水检测中的应用

为快速高效地检测人星状病毒,建立了一种新的试验方法——环介导等温扩增技术(loop-mediated isothermal amplification technology,LAMP),并对人星状病毒LAMP反应条件进行了优化.结果表明,镁离子终浓度为4 mmol·L-1、甜菜碱终浓度为1mol.L-1的25μL体系下...     

Quantification of copepod gut content by differential length amplification quantitative PCR (dla-qPCR)

Quantification of feeding rates and selectivity of zooplankton is vital for understanding the mechanisms structuring marine ecosystems. However, methodological limitations have made many of these studies difficult. Recently, molecular based methods have demonstrated that DNA from prey species can be used to identify zooplankton gut contents, and further, quantitative gut content estimates by quantitative PCR (qPCR) assays targeted to the 18S rRNA gene have been used to estimate feeding rates in appendicularians and copepods. However, while standard single primer based qPCR assays were quantitative for the filter feeding appendicularian Oikopleura dioica, feeding rates were consistently underestimated in the copepod Calanus finmarchicus. In this study, we test the hypothesis that prey DNA is rapidly digested after ingestion by copepods and describe a qPCR-based assay, differential length amplification qPCR (dla-qPCR), to account for DNA digestion. The assay utilizes multiple primer sets that amplify different sized fragments of the prey 18S rRNA gene and, based on the differential amplification of these fragments, the degree of digestion is estimated and corrected for. Application of this approach to C. finmarchicus fed Rhodomonas marina significantly improved quantitative feeding estimates compared to standard qPCR. The development of dla-qPCR represents a significant advancement towards a quantitative method for assessing in situ copepod feeding rates without involving cultivation-based manipulation.     

Brevetoxins in sharks and rays (Chondrichthyes, Elasmobranchii) from Florida coastal waters

In October 2000, a mass mortality of blacktip sharks (Carcharhinus limbatus) and Atlantic sharpnose sharks (Rhizoprionodon terraenovae) in northwest Florida occurred in conjunction with a Karenia brevis red tide bloom. Before this incident, no information existed on red tide-induced shark mortalities or baseline brevetoxin levels in sharks and rays from red tide-endemic areas. We report here that brevetoxin accumulation in live and red tide-killed elasmobranchs is common during K. brevis blooms and non-bloom periods. Strong relationships were found between the frequency of red tide blooms and the average brevetoxin concentrations in elasmobranch tissues. The presence of brevetoxins in Atlantic coast sharks in the absence of documented K. brevis blooms may suggest that blooms are occurring in areas that are not well monitored. Although red tide-related shark mortalities are rarely observed, the presence of brevetoxins in shark embryos raises questions about the effects these toxins may have on the reproductive success of sharks.     

Brevetoxin exposure in bottlenose dolphins (Tursiops truncatus) associated with Karenia brevis blooms in Sarasota Bay, Florida

Bottlenose dolphins (Tursiops truncatus) face a variety of threats, including risk of exposure to brevetoxins produced by blooms of the harmful alga Karenia brevis. This study investigated brevetoxin exposure in a population of dolphins inhabiting Sarasota Bay, Florida, USA (27°N, 82°W), utilizing tissues from dolphins recovered between 1994 and 2003. Brevetoxin levels detected by ELISA in tissues, gastric samples and excreta from dolphin carcasses (n = 19) associated with K. brevis blooms were compared with with levels in carcasses (n = 16) associated with background K. brevis conditions. In the K. brevis-exposed set, 84% of dolphin carcasses recovered during K. brevis blooms had detectable brevetoxin levels, with values ranging between 7 and 2,896 ng PbTx-3 eq g⁻¹. Over 50% of dolphin carcasses recovered during non-bloom conditions also tested positive by ELISA for brevetoxins, with concentrations ranging from 6 to 44 ng PbTx-3 eq g⁻¹. Control samples from the east coast of Florida were negative by the ELISA. Results from this study establish baseline brevetoxin body burdens in a dolphin population frequently exposed to K. brevis blooms, and data indicate that dolphin carcasses not associated with large-scale mortality events can contain levels of brevetoxins comparable to carcasses stranding during such events.     

Transfer of brevetoxins to a tellinid bivalve by suspension- and deposit-feeding and its implications for clay mitigation of <Emphasis Type="Italic">Karenia brevis</Emphasis> blooms

Blooms of the brevetoxin-producing Karenia brevis in the Gulf of Mexico cause massive fish kills, food poisoning and adverse respiratory effects in humans. Sedimentation of toxic cells following inert clay application could reduce toxin incorporation by commercially important suspension-feeding bivalves and thus prevent direct public health impacts, but could potentially lead to brevetoxin (PbTx) accumulation by benthic deposit-feeders. The goal of this study was therefore to compare suspension- and deposit-feeding as pathways for brevetoxins. We investigated: (1) the effect of toxic K. brevis on both feeding modes using a facultative deposit-suspension feeding tellinid bivalve, the clam Macoma balthica, as a model species and (2) the relative effectiveness of brevetoxin transfer via suspension- and deposit-feeding over 24-h exposure. Sedimentation of K. brevis was achieved by treatment with 0.25 g phosphatic clay l⁻¹ and brevetoxin concentrations were measured by ELISA. Karenia brevis reduced both suspension- and deposit-feeding activity. This study demonstrates that brevetoxins can be rapidly accumulated by a surface deposit-feeding bivalve from sedimented K. brevis cells and that comparable toxin levels can be attained by both suspension- and deposit-feeding modes [1.2–1.6 μg PbTx (g tissue wet weight)⁻¹]. Deposit-feeding clams generally do not pose a direct threat to humans but may provide a pathway for brevetoxin food web transfer.     

原废水培养基分离活性污泥中的苯酚降解细菌

用未经处理的含酚焦化工业废水为基础配制培养基，采用平板涂布法，在10^-5稀释度下得到100个单菌落分离物．它们均能在以苯酚(500-800mg L^-1)为唯一碳源的无机盐培养基上生长，其16S rDNA基因的ARDBA多态性分析将这些分离物划分成4个分类操作单元(OTUs)．各单元代表株的16S rRNA基因序列分析和检索结果表明，97个分离物与Alcaligenes faecalis同源性达99％，2个分离物分别与Arthrobacter nicotianae和Klebsiella sp.99％同源．另一个分离物与Ochrobactrum sp．96％同源．苯酚经化酶大亚基基因(LmPHs)PCR扩增表明，除类似Arthrobacter nicotianae的菌株外，其它3个分类操作单元的降酚菌都能利用多组分苯酚经化酶代谢途径进行酚代谢．图2表2参11     

重金属Cd、Pb对褐菖鲉肝脏组织中HSPs基因表达的影响

为了解重金属污染对海洋鱼类热休克蛋白(HSPs)基因表达的影响,将褐菖鲉(Sebastiscus marmoratus)分别暴露于1.6、8、40、200、500μg·L~(-1)Cd、Pb溶液中,用环介导等温扩增技术(loop-mediated isothermal amplification,LAM P)定量检测褐菖鲉肝脏HSP60、HSP70、HSP90、HSC70 mRNA表达量。结果表明:Pb仅在40μg·L-1时显著抑制HSP60、HSP90、HSC70 mRNA表达量,8μg·L~(-1)时即可显著抑制HSP70 m RNA表达量,并在40μg·L~(-1)时达到最小值;Cd对HSP60、HSP90、HSC70的诱导不明显,但能显著诱导HSP70,并在500μg·L~(-1)时达到最大值。相比之下,褐菖鲉肝HSP70基因对重金属Cd、Pb污染较为敏感,有潜力成为监测海洋重金属污染的预警分子。     

朱■的随机扩增多态DNA分析与种内亲缘关系研究

利用随机扩增多态ＤＮＡ技术对朱（Ｎｉｐｐｏｎｉａｎｉｐｐｏｎ）８个个体进行了随机扩增多态ＤＮＡ分析．用２０个１０ｂｐ的随机引物对每只朱的基因组ＤＮＡ进行扩增,共得到１６８个扩增片段,其中共有片段为１０２个．根据聚类分析所得到的树状图确定了８只朱的亲缘关系,这为进一步构建全部朱个体的谱系关系图打下了基础,有利于制定更有效的朱保护计划     

Use of sodium transfer tissue biosensor (STTB) for monitoring of marine toxic organism

A highly sensitive sodium (Na+) transfer tissue biosensor (STTB) was designed using a frog bladder membrane to measure paralytic shellfish poisons (PSP). The STTB consists, of a Na+ electrode covered by the membrane, which was then integrated into a flow-through system for continuous measurements. In the absence of Na+ channel blocker, active transfer of Na+ occurred from inside to outside across the frog membrane. When the STTB was used to measure the Na+ -dependent dissociation of PSP, it was able to detect PSB at a level contained in a single cell. However, 5 fg or higher (100 cells or more) is needed for accurate and reproducible measurements. The toxicity obtained by the STTB was significantly correlated (r = 0.9449) to that determined by the HPLC. Therefore, the simple method of the STTB can be used not only to detect a low level PSP in toxic plankton populations, but also to monitor poisons in shellfish.     

朱鹮的随机扩增多态DNA分析与种内亲缘关系研究

利用随机扩增多态DNA技术对朱鹮 ( Nipponia nippon ) 8个个体进行了随机扩增多态DNA分析. 用20个10bp的随机引物对每只朱鹮的基因组DNA进行扩增,共得到168个扩增片段,其中共有片段为102个. 根据聚类分析所得到的树状图确定了8只朱鹮的亲缘关系,这为进一步构建全部朱鹮个体的谱系关系图打下了基础,有利于制定更有效的朱鹮保护计划.