重金属Zn^(2+)胁迫下米氏凯伦藻(Karenia mikimotoi)的生长生理响应研究

采用室内培养试验方法,以米氏凯伦藻(Karenia mikimotoi)为生物材料,设置不同浓度梯度的重金属Zn^(2+)(0、1、5、10、15和20mg·L^(-1)),主要测定藻细胞密度、光合色素、光合效率、抗氧化酶类、丙二醛(malondialdehyde,MDA)等相关指标,探讨重金属Zn^(2+)胁迫下米氏凯伦藻(Karenia mikimotoi)的生长和生理特征。结果发现,在1和5 mg·L^(-1)的Zn^(2+)浓度下,米氏凯伦藻细胞依然保持较好生长繁殖,表明其具有一定的耐受性,而随着重金属Zn^(2+)浓度的提高,细胞生长受到毒害抑制。光合色素含量呈现动态变化,试验结束时(96 h)叶绿素a、叶绿素b和胡萝卜素含量有各自不同的变化趋势,最大光能转化效率(Fv/Fm)表现出先升后降的趋势。Zn^(2+)浓度为10mg·L^(-1)时,超氧化物歧化酶(superoxidedismutase,SOD)活性显著高于对照;过氧化氢酶(catalase,CAT)活性呈现出先上升后下降的趋势,且5、10、15和20mg·L^(-1)Zn^(2+)浓度下米氏凯伦藻的过氧化氢酶(CAT)活性均显著高于对照;10、15mg·L^(-1)Zn^(2+)浓度下米氏凯伦藻的总抗氧化能力(total antioxidantcapacity,T-AOC)显著高于对照,而20mg·L^(-1) Zn^(2+)浓度下的显著低于对照。丙二醛(MDA)呈现出随着Zn^(2+)浓度提高而增加的趋势,5、10、15和20mg·L^(-1)Zn^(2+)浓度下米氏凯伦藻的丙二醛(MDA)均显著高于对照。结果可为了解重金属对海洋微藻的毒性作用提供数据参考。

我国近海米氏凯伦藻(Karenia mikimotoi)藻华发生概况、危害及其生态学机制

米氏凯伦藻(Karenia mikimotoi)隶属于裸甲藻目(Gymnodiniales)凯伦藻科(Kareniaceae)。该藻广泛分布于全球海域,是海洋中的主要有毒有害赤潮藻,也是我国第二大灾害性赤潮肇事种。米氏凯伦藻可产生半乳糖脂、溶血毒素、鱼毒素和部分细胞毒素,当其大规模暴发时会造成严重的生态灾害和经济损失,并危及海产品安全和人类健康。该藻为广温广盐种,光照耐受阈宽,多以光合作用营自养生长,主要繁殖方式为无性繁殖;另外,米氏凯伦藻能吸收利用不同形态的磷盐,并可进行吞噬营养。这些生态学特性使其具有环境适应优势,在适宜条件下过度繁殖引发藻华。

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 nega- tive 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 tak- en 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 tech- niques 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 differ- ence （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.

Microbial community coexisting with harmful alga Karenia mikimotoi and microbial control of algal bloom in laboratory

Algicidal bacteria have been frequently isolated from algal blooming areas.However,knowledge regarding the microbial communities coexisting with microalgae and their potential application in preventing harmful algal blooms(HABs)is limited.In this study,we investigated the composition of the microbial community coexisting with harmful alga Karenia mikimotoi and its responses to algal control via nutrient stimulation or by adding algicidal strain in microcosms.The microorganisms inhabiting the K.mikimotoi culture consisted of 24 identifi ed phyla,including dominant Proteobacteria(relative abundance 76.24%±7.28%)and Bacteroidetes(22.67%±8.32%).Rhodobacteraceae,Phaeodactylibacter,and Maritimibacter predominated during the algal cultivation.Both the added nutrient and fermentation broth of algicidal strain Pseudoalteromonas QF1 caused a massive death of K.mikimotoi and substantial changes in the coexisting microbial community,in which Rhodobacteraceae and Phaeodactylibacter signifi cantly decreased,while Halomonas and Alteromonas increased.Core operational taxonomic units(OTUs)analysis indicated that 13 OTUs belonging to Rhodobacteraceae,Maritimibacter,Marivita,Nisaea,Phaeodactylibacter,Citreicella,Halomonas,Alteromonas,Marinobacter,Muricauda,and Pseudoalteromonas dominated the changes of the microbial communities observed in the K.mikimotoi culture with or without treatments.Collectively,this study indicated that microbial community inhabiting K.mikimotoi culture includes potential algicidal bacteria,and improves our knowledge about microbial community succession during biocontrol of K.mikimotoi via nutrient stimulation or by adding isolated algicidal strains.

The First Occurrence of Red Tide Caused by Karenia sp.in the Atlantic Moroccan Coast(Oualidia Lagoon)

An intense bloom of Karenia sp.was reported in the Oualidia lagoon,part of Atlantic Moroccan coast.The highest concentrations are 1.04×10^7 cells/L,and have been noted at the park station 7.High nutrient concentrations have been observed,and PO4 was the highest value(av.396.18μg/L)recorded at Parc 7.This massive proliferation caused a red tide which extended over 25 km from the Atlantic coast.This event was accompanied by stranding of macroalgae.

温度对旋链角毛藻(Chaetoceros curvisetus)和米氏凯伦藻(Karenia mikimotoi)生长及硝酸还原酶活力的影响

选用东海常见的两种赤潮肇事藻种：旋链角毛藻和米氏凯伦藻,采用一次性培养实验,研究了不同温度对两种赤潮藻生长及硝酸还原酶活力（NRA）的影响.研究结果表明,在10℃-30℃条件下旋链角毛藻均能正常生长,且生长曲线均符合S-logistic2种群增长模型;而米氏凯伦藻在10℃和30℃条件下不能正常生长,在其他温度条件下生长情况与旋链角毛藻相似.温度适宜时,两种藻的硝酸还原酶活力最大值（NRAmax）、最大生长速率（μmax）和终止生物量（Bf）随温度的变化趋势基本一致,说明温度的高低可通过影响细胞硝酸还原酶活力大小间接影响藻类的生长.旋链角毛藻单位体积的NRAmax和最大生长速率均大于米氏凯伦藻,说明旋链角毛藻能够更好地吸收利用硝酸盐.

甲藻(Dinophyta)凯伦藻科(Kareniaceae)的分类学研究与展望

凯伦藻科(Kareniaceae)是甲藻门中较新建立的一个科。凯伦藻科种类广泛分布于全球海域,目前已报道3属30个种。由于该科大部分种类频发藻华,严重威胁近海生态安全和水产养殖,因而备受关注。自20世纪90年代起,我国沿岸也逐渐成为凯伦藻藻华的重灾区,凯伦藻曾引发我国最严重的几次藻华事件。本文对本科种类分类学特征进行详细描述,翻译其中文名,编制属、种分类检索表,以期为我国凯伦藻生物多样性研究以及凯伦藻藻华防治提供基础而必要的种源信息。

福建平潭海域米氏凯伦藻(Karenia mikimotoi)增殖影响因子研究

分析了2018年5-6月平潭海域的气象及水文情况,探讨了环境因子对米氏凯伦藻(Karenia mikimotoi)增殖的影响.Spearman相关性分析显示,米氏凯伦藻细胞密度与气温、水温、叶绿素a呈极显著正相关,与盐度、压强呈极显著负相关.平潭海域米氏凯伦藻增殖的适宜盐度为28.4~30.0,水温为23.5~25.0℃,压强为99.7~101.0 kPa,叶绿素a为1.5~7.2μg·L^-1,其最高密度8.5×10^5 cells发生在海水盐度为28.9,水温为24.6℃,压强为99.9 kPa.平潭海域浮游植物4-5月以中肋骨条藻为优势种,5-6月演替为米氏凯伦藻为优势种,温度、盐度和气压是影响其增殖的重要环境因子.

Effects of co-existing microalgae and grazers on the production of hemolytic toxins in Karenia mikimotoi

Karenia mikimotoi (Miyake & Kominami ex Oda) Hansen & Moestrup is associated with harmful algal blooms in temperate and subtropical zones of the world. The hemolytic substances produced by K. mikimotoi are thought to cause mortality in fishes and invertebrates. We evaluated the composition of the hemolytic toxin produced by K. mikimotoi cultured in the laboratory using thin-layer chromatography.In addition, we evaluated the effect of co-occuring algae (Prorocentrum donghaiense and Alexandrium tamarense) and the cladoceran grazer Moina mongolica on hemolytic toxin production in K. mikimotoi. The hemolytic toxins from K. mikimotoi were a mixture of 2 liposaccharides and 1 lipid. Waterborne clues from P. donghaiense and A. tamarense inhibited the growth of K. mikimotoi but increased the production of hemolytic toxins. Conversely, K. mikimotoi strongly inhibited the growth of caged P. donghaiense and A. tamarense. In addition, the ingestion of K. mikimotoi by M. mongolica induced the production of hemolytic toxins in K. mikimotoi. Taken together, our results suggest that the presence of other microalgae and grazers may be as important as environmental factors for controlling the production of hemolytic substances. K. mikimotoi secreted allelochemicals other than unstable fatty acids with hemolytic activity. The production of hemolytic toxins in dinoflagellates was not only dependent on resource availability, but also on the risk of predation. Hemolytic toxins likely play an important role as chemical deterrents secreted by K. mikimotoi.

Allelopathic interactions between Prorocentrum micans and Skeletonema costatum or Karenia mikimotoi in laboratory cultures

Algal allelopathy is an ecological/physiological phenomenon that has focused attention on the interactions among algae and the production of algal toxins. We investigated the allelopathic interactions between the dinoflagellate genus Prorocentrum micans and diatom genus Skeletonema costatum and between P. micans and dinoflagellate genus Karenia mikimotoi using bi-algal cultures. Because the effects were species-specific and size-dependent, we evaluated the effect of different initial densities. At low densities of P. micans and high densities of S. costatum inoculated into the same medium, the growth of P. micans was weakly restrained, whereas the growth of S. costatum was significantly suppressed. S. costatum and K. mikimotoi were strongly inhibited by P. micans, in both the bi-algal cultures and enriched filtrates. Direct cell-to-cell contact was not necessary to gain a competitive advantage, thus, our results suggest that P. micans inhibited the growth of S. costatum and K. mikimotoi by the release of allelochemical(s). Last, a mathematical model was used to simulate growth and interactions between P. micans and S. costatum and between P. micans and K. mikimotoi in bi-algal cultures.

Toxic effects of Karenia mikimotoi extracts on mammalian cells

Karenia is one of the most harmful and representative red tide genus in a temperate zone. Blooms caused by this genus have resulted in massive fish death in the South China Sea and the East China Sea. However, the potential effects of this dinoflagellate on human health through the transfer of toxins via marine food webs, and the mechanisms of toxicity, are still unknown. Therefore, we examined the toxic effects of a strain of K. mikimotoi (isolated from the South China Sea) on the proliferation and morphology of four mammalian cell lines (two normal cell lines and two cancer cell lines). In addition, we carried out a preliminary investigation on the mechanism of toxicity of the alga. The results show that the polar lipid-soluble component of K. mikimotoi significantly inhibited proliferation of the four cell lines, and resulted in the cells becoming spherical, swollen and damaged. The result of Annexin V and PI double-staining confirmed that cell membranes were disrupted. The malonaldehyde (MDA) contents in the medium of the four cell lines treated with the polar-lipid extracts all increased significantly, which indicates that the polar-lipid toxins produced by K. mikimotoi could adversely affect mammalian cells by inducing lipid peroxidation. We conclude that K. mikimotoi is a potential threat to human health, and the comprehensive effect of this dinoflagellate and its mechanisms should be investigated further.

Interspecific competition and allelopathic interaction between Karenia mikimotoi and Dunaliella salina in laboratory culture

Algal allelopathy is a manifold ecological/physiological phenomenon that is focused on chemical interactions and autotoxicity. We investigated the allelopathic interactions between Karenia mikimotoi and Dunaliella salina in laboratory cultures based on diff erent temperature(15°C, 20°C, and 25°C) and lighting(40, 80, and 160 μmol/(m^2 ·s)) conditions. The growth of D. salina in bi-algae culture(1:1 size/density) was significantly restrained. The results of cell-free filtrate culture indicate that direct cell-tocell contact was not necessary in interspecific competition. Further experimental results demonstrated that allelochemicals released from K. mikimotoi were markedly influenced by both temperature( P =0.013) and irradiance( P =0.003), resulting in diff erent growth characteristics of D. salina in filtrate mediums. Compared with the plateau period, K. mikimotoi exudates in the exponential phase had a stronger short-term inhibition eff ect on D. salina in normal conditions. A clear concentration-dependent relationship was observed in the eff ect of allelochemicals released from K. mikimotoi with low-promoting and high-repressing eff ects on D. Salina in a short time-scale. In addition, allelopathic substances remain stable and eff ective under high temperature and pressure stress. Many flocculent sediments adhering with D. salina cells were observed in all filtrate mediums, while the quantity and color depended on the original culture conditions.

Will harmful dinoflagellate Karenia mikimotoi grow phagotrophically?

We studied the phagotrophic ability of dinoflagellate strain Karenia mikimotoi KM-Lü (isolated from the South China Sea), using fluorescent microspheres, bacteria isolated from the culture of K. mikimotoi and a marine microalgae Isochrysis galbana. We found that K. mikimotoi cultured under conditions of high light intensity could ingest fluorescent microspheres (diameters 0.5 and 2.0 μm) and fluorescence-labeled bacteria and microalgae. Under a low light intensity, however, only fluorescent microspheres (diameter 0.5 μm) and fluorescence-labeled microalgae were ingested. K. mikimotoi showed better growth by ingesting living marine bacteria or microalgae I. galbana than the controls, either in nutrient-depleted or nutrient-replete conditions. In nutrient-depleted conditions, the growth of K. mikimotoi was more significant with I. galbana as the prey item. In conclusion, the harmful dinoflagellate K. mikimotoi from the South China Sea has apparent phagotrophic ability, and some marine bacteria and microalgae may promote the growth of K. mikimotoi.

黏土组合芽孢杆菌Ba3控制赤潮甲藻研究

基于细菌溶藻和黏土吸附的特性,探索了溶藻菌Ba3与黏土组合控藻技术。正交实验确定了投加量为1.0%Ba3无菌滤液和100 mg·L^(-1)黏土为最佳组合,该组合技术在24 h内对米氏凯伦藻的溶藻率达到94.0%。分别对比了单独投加无菌滤液、黏土、黏土+Ba3无菌滤液、黏土+PAC、黏土+H_(2)O_(2)的控藻效果。结果显示,黏土+PAC、黏土+H_(2)O_(2)组合在12 h后出现了藻密度“回弹”现象,黏土+Ba3无菌滤液在48 h后的溶藻效果达到95%以上显著高于其他组合,检测试验前后叶绿素a浓度的变化,进一步验证了Ba3无菌滤液组合黏土的高效控藻效果,有望在赤潮甲藻控制中应用推广。

赤潮藻米氏凯伦藻(Karenia mikimotoi)藻际细菌群落及其原位抑藻效应

采用16S rRNA高通量测序技术分析了赤潮藻米氏凯伦藻藻际细菌群落组成,探究了原位营养刺激下藻际细菌对米氏凯伦藻生长的抑制作用。结果表明:米氏凯伦藻藻际环境以Proteobacteria为主(α-Proteobacteria 48.1%,γ-Proteobacteria 8.6%),其次Sphingobacteria 22.4%,Cyanobacteria 10.8%。通过加混合抗生素获得了米氏凯伦藻无菌培养体系,验证了所加营养液对藻的生长无明显影响。向米氏凯伦藻培养体系中添加体积比5%的2216E培养基、甘油和柠檬酸钠时,米氏凯伦藻的生长受到显著抑制,抑制率分别为99.6%、96.0%和91.5%。对2216E刺激下藻际细菌群落变化进行分析表明,α-Proteobacteria丰度增至58.7%,其中Rhodobacteraceae由8.7%增至22.3%,Thalassobaculum由2.7%增至13.3%,γ-Proteobacteria增至13.3%,其中Pseudomonas由2.4%增至4.9%。米氏凯伦藻藻际环境存在着丰富的微生物类群,适量外部营养刺激可以实现藻际细菌原位抑藻效应。

pH对米氏凯伦藻(Karenia mikimotoi)种群生长、营养吸收及无机碳亲和力的影响

随着海水酸化以及沿海赤潮爆发诱发水体p H的上升,p H对海洋藻类生理生态影响逐渐为人类所关注。本实验以东海常见赤潮藻——米氏凯伦藻(Karenia mikimotoi)为研究对象,研究不同p H对其生理状态的影响。结果显示,p H对米氏凯伦藻的生长、营养吸收、叶绿素、光合速率以及对无机碳的亲和力存在显著影响。当p H=6.2时,米氏凯伦藻的生长受到抑制,细胞生长速率,营养盐吸收率,最大光合速率以及对无机碳的亲和力都最低;当p H=8.2时,细胞的生长最快,对营养盐的吸收也最快而且最大光合速率达到最高;p H=7.2和9.2时,藻细胞能维持较高的生长,且对无机碳的亲和力高于p H=8.2实验组。总而言之,过高或过低的p H胁迫会引起米氏凯伦藻生理状态的不良变化,尤其当p H过低时米氏凯伦藻生理状态受到严重影响。

有害甲藻米氏凯伦藻(Karenia mikimotoi)对海洋青鳉鱼(Oryzias melastigm)的急性毒性效应研究

米氏凯伦藻(Karenia mikimotoi)是我国近岸海域常见的有毒有害藻华肇事种,能产生多种毒素,其发生藻华时会使大量鱼类或贝类死亡。本研究从细胞生物学、氧化和免疫酶活性角度研究米氏凯伦藻对海洋青鳉鱼(Oryzias melastigma)的急性毒性效应(72 h),以期揭示米氏凯伦藻对鱼类的致死原因。组织切片结果表明,高浓度的米氏凯伦藻[(1.37~1.52)×10^(7)cells/L]可以导致海洋青鳉鱼鳃丝断裂,成团状与相邻鳃丝融合,鳃丝上皮细胞的细胞核发生偏移,并使肝细胞出现空泡化现象。高浓度的米氏凯伦藻能够抑制海洋青鳉鱼的鳃和肝组织的SOD酶活性和CAT酶活性,引起海洋青鳉鱼脂质过氧化,导致丙二醛(MDA)含量升高,对组织造成损伤。本研究结果表明,米氏凯伦藻可能通过氧化应激反应使海洋青鳉鱼的鳃和肝组织受损,为深入阐明米氏凯伦藻的毒性机制提供参考。

陆源DON亲水和疏水组分对米氏凯伦藻和中肋骨条藻生长及种间竞争的影响

本文以米氏凯伦藻(Karenia mikimotoi)和中肋骨条藻(Skeletonema costatum)为研究对象,采用畜禽养殖源有机质为DON氮源,应用XAD-8树脂分离亲水性和疏水性组分,并进行单一藻和双藻混合DON加富室内受控培养实验。结果表明,亲水性DON组分和疏水性DON组分均可促进米氏凯伦藻和中肋骨条藻的生长,其中,亲水组分的促进作用优于疏水组分(P<0.05,α=0.05)。在溶解无机氮浓度不受限条件下,通过DON加富培养,中肋骨条藻相对于米氏凯伦藻具有竞争优势,表现为氮营养盐吸收速率常数、氮亲和力指数和浮游植物生长速率常数均大于米氏凯伦藻(P<0.05,α=0.05),这可能与米氏凯伦藻在吸收利用DON过程中,需要分泌更多的亮氨酸氨肽酶以提高其营养供应策略有关,而中肋骨条藻则可以在较低的酶活性下吸收利用不同形态和组成的氮。本文研究结果有助于从动力学上加深不同DON组分对长江口赤潮多发区中肋骨条藻赤潮爆发支撑作用的理解。

米氏凯伦藻对海水青鳉影响效应的转录组学分析

近年来我国近岸米氏凯伦藻(Karenia mikimotoi)藻华频发,给当地水产养殖业带来严重的损失,但该藻在海洋生物分子水平上的影响尚不明确。本实验通过转录组学分析,探究了米氏凯伦藻对模式生物海水青鳉(Oryzias melastigma)鳃和肝脏mRNA转录水平的影响。结果发现受藻作用96 h后,青鳉鳃与肝脏中分别有508个与604个差异表达基因(differentially expressed genes,DEGs),其中上调基因分别有184个和390个,下调基因分别有324个和214个。Gene ontology(GO)功能分类结果显示DEGs多集中于生物进程和分子功能,进一步GO富集分析结果表明,米氏凯伦藻能够显著影响青鳉鳃组织中凝血酶激活受体活性、受体信号通路和离子跨膜运输等相关通路;肝脏中氧运输和结合进程相关通路也受到显著影响。Kyoto encyclopedia of genes and genomes(KEGG)功能富集分析表明鳃和肝脏组织中DEGs均显著富集于免疫系统的补体和凝血级联反应,鳃DEGs还富集在IL-17免疫信号分子和细胞因子相互作用信号转导通路;而肝脏DEGs还富集在脂质、氨基酸代谢通路。此外,免疫因子serpine1、hsp90b1、bcl2l1在鳃中均被显著抑制,而凝血因子f2、f5、plg在肝脏中均被显著上调。实验结果表明米氏凯伦藻可能对海水青鳉鳃造成了一定程度的氧化损伤,也可能通过激活IL-17信号通路导致免疫炎症的发生。同时该藻还导致海水青鳉肝脏纤溶系统被激活,代谢功能发生变化。

米氏凯伦藻对褶皱臂尾轮虫、卤虫和黑褐新糠虾存活的影响

初步研究了一株米氏凯伦藻对褶皱臂尾轮虫(Brachionus plicatilis)、卤虫(Artemia salina)幼体和黑褐新糠虾(Neomysis awatschensis)的毒性效应和机制。研究发现米氏凯伦藻(Karenia mikimotoi)在较低密度下就能明显减少轮虫的种群数量,24hEC50仅为20个/mL左右;该藻的各组分毒性比较结果显示,只有藻液和细胞重悬液有这种毒害作用,而去藻过滤液和藻细胞破碎液的影响不明显,表明这种毒害作用可能是由于活的藻细胞引起的;在米氏凯伦藻中卤虫和黑褐新糠虾的存活数量也明显下降。结果表明,米氏凯伦藻赤潮可能通过直接影响存活从而对浮游动物产生影响。