一株抑制浒苔生长海洋细菌的分离与鉴定

从青岛近海海泥中分离了57 株细菌, 通过毒性实验, 得到了一株对浒苔具有较强毒性的细菌EP23。对该菌株进行了菌落、菌体形态学观察、生理生化特征分析及16S rDNA 序列分析, 鉴定该菌为弯曲芽孢杆菌（Bacillus flexus）。该菌培养液高速离心的上清液对浒苔具有较强毒性, 表明弯曲芽孢杆菌EP23 是通过...     

河流弧菌对牙鲆表皮黏液黏附作用的研究

为了解河流弧菌对牙鲆表皮黏液的黏附特性,采用3H-TdR同位素示踪方法研究了细菌浓度、孵育时间、孵育温度、pH值、阳离子、碳水化合物等因子对河流弧菌黏附作用的影响。试验结果表明:河流弧菌能很好地黏附于牙鲆表皮黏液,黏附量随菌浓度升高而增大并符合饱和黏附动力学:y=417.89ln(x)+691.57(R2=0.986);在30℃下黏附量随着孵育时间的延长而增加,180min趋于饱和;孵育温度在4~30℃范围内黏附量随温度升高而增多,30℃时黏附量最大;pH值酸性较碱性黏附作用明显,pH为5时最强;NaCl浓度在0.5%~4.5%范围内,细菌黏附作用随着浓度升高而增强,较纯水存在显著性差异(P<0.05);Ca2+,Mg2+能显著加强河流弧菌的黏附作用,但Ca2+作用明显超过Mg2+;葡萄糖等8种碳水化合物能显著促进河流弧菌的黏附作用(P<0.05)。以上结果说明:河流弧菌对牙鲆表皮黏液的黏附量较大,这有利于进一步感染、致病;河流弧菌的黏附作用受温度、pH值、阳离子、碳水化合物等多种环境因子的影响,较高的水温有利于河流弧菌的黏附,有利于疾病的发生。     

双水相萃取法富集分离螺旋藻藻蓝蛋白的研究

以PEG／硫酸钠双水相体系,经一次萃取从钝顶螺旋藻（Spirulina platensis）细胞破碎液中富集分离藻蓝蛋白。结果表明,萃取最适宜的条件为12％PEG4000,15％Na2SO4,1％KCl,藻蓝蛋白收率为91．2％,分配系数达到8．01,分离因数达到6．33。对于螺旋藻藻蓝蛋白的富集分离,双水相萃取法与传统的盐析沉淀法相比,具有节省操作时间、简化操作过程、降低能耗和成本以及易于工艺放大等优点。     

病原性溶藻弧菌对大黄鱼鳃黏液黏附作用研究

采用3H-TdR同位素示踪方法研究了溶藻弧菌对大黄鱼鳃黏液黏附作用.结果表明,溶藻弧菌的黏附量随着菌浓度的增加而升高并在180 min后达到饱和;在温度15~30℃、pH偏酸条件下黏附作用较强,Na+是溶藻弧菌黏附作用必需的,Ca2+起着辅助作用;溶藻弧菌经营养饥饿、热处理及高碘酸处理后黏附作用有明显下降;8种碳水化合物都有不同程度的促进溶藻弧菌的黏附作用,其中葡萄糖、甘露糖、果糖及麦芽糖有明显促进作用.研究结果表明:海水中的溶藻弧菌可很好地黏附于大黄鱼鳃黏液,其黏附作用受环境因子影响,并且与溶藻弧菌的表面碳水化合物、热敏结构等有着密切关系.     

浒苔孢子在不同材料基质上的附着萌发差异性研究

本研究针对黄海绿潮早期发生过程,以浒苔生殖细胞附着生长特征为切入点,以其主要原因藻种浒苔(Ulva prolifera)为材料,对比研究了不同材料附着基质对浒苔孢子附着萌发的影响。发现孢子在2—6h内的附着率达到80%,尤其是在前2h内的附着速率最快,而且在不同材料的基质上都能完成附着萌发,但萌发数量存在显著差异性,孢子在紫菜养殖筏架相关材料(塑料、竹片、尼龙网衣、塑料网衣)上的萌发率显著高于浅滩、礁石和养殖池相关材料(细砂、海泥、石块)。浒苔孢子还可以在藻段上完成附着萌发,而橡胶和鱼粉袋相关材料对浒苔孢子的萌发存在抑制作用。对比浒苔孢子和配子的萌发特性发现两者有异同点,两者在塑料、竹片等筏架相关材料上萌发率均较高,而孢子在藻段以及橡胶浸出液和鱼粉溶液中的萌发数均显著高于配子。本研究发现紫菜养殖筏架更有利于浒苔孢子的附着萌发,这进一步说明苏北浅滩数十万亩的紫菜养殖筏架在绿潮早期形成过程中为浒苔孢子提供了重要的附着基质。本研究结果将为探讨紫菜养殖筏架在绿潮早期发生过程中的重要作用,并为养殖筏架防附着材料的选用提供参考。     

Adhesion of pathogenic Vibrio alginolyticus to the gill mucus of Pseudosciaena crocea

Adhesion of Vibrio alginolyticus to the gill mucus of Pseudosciaena crocea has been investigated using [methyl—3H]thymidine as isotope tracer. The results showed that: the adhesive quantity of V. alginolyticus increased with bacterial concentrations and reached equilibrium after incubated for 180 min; the higher adhesive quantity was obtained at 15~30 ℃ and sourish conditions; adhesion of V. alginolyticus could not achieved without Na , and Ca2 played an auxiliary role in the bacterial adhesion; adhesion of V. alginolyticus was inhibited remarkably by starvation, heat treatment and periodic acid treatment; all of the eight kinds of carbohydrates investigated enhanced the adhesion of V. alginolyticus to the gill mucus of P. crocea, among them, glucose, mannose, fructose and maltose showed the specially enhanced adhesion. The results indicated that V. alginolyticus could adhere to the gill mucus of P. crocea facilely in seawater, and this bacterial adhesion was influenced by environmental factors and closely related to superficial carbohydrate structures and some heat-sensitive structures.     

熊本牡蛎、葡萄牙牡蛎和长牡蛎组织菌群构成及功能的比较分析

为探究健康二龄熊本牡蛎、葡萄牙牡蛎和长牡蛎5种组织间细菌群落组成、多样性和功能的差异,本研究利用Illumina高通量测序技术和PICRUSt2对3种牡蛎5种组织中的细菌群落构成及其潜在功能进行了比较分析。结果显示,在3种牡蛎的5种组织中共鉴定到6 020个细菌OTUs,其中3种牡蛎整体间共有的OTUs数占42.4%。3种牡蛎5种组织中的优势菌主要隶属于γ-变形菌纲（γ-proteobacteria）、α-变形菌纲（α-proteobacteria）、柔壁菌门（Tenericutes）和厚壁菌门（Firmicutes）。弧菌属（Vibrio）在熊本牡蛎的鳃、外套膜、性腺和血淋巴中的相对丰度均显著高于长牡蛎和葡萄牙牡蛎,且分别在熊本牡蛎外套膜、长牡蛎性腺和葡萄牙牡蛎血淋巴中相对丰度最高,但均在3种牡蛎肝胰腺中相对丰度最低。3种牡蛎5种组织间的菌群多样性均有所差异,其中在熊本牡蛎鳃、葡萄牙牡蛎外套膜和长牡蛎血淋巴中菌群多样性最高,而在熊本牡蛎血淋巴、葡萄牙牡蛎性腺和长牡蛎肝胰腺中菌群多样性最低。在不区分组织的情况下,3种牡蛎整体间的菌群结构存在显著差异（r=0.661,p<0.001）。此外,在熊本牡蛎肝胰腺、葡萄牙牡蛎鳃和长牡蛎血淋巴中细菌参与的能量代谢相关功能通路的相关丰度显著高于对应牡蛎的其他组织。本文结果表明,熊本牡蛎、葡萄牙牡蛎和长牡蛎组织内的菌群构成存在差异,且由细菌介导的功能也随牡蛎种类和组织类型发生改变。     

海假交替单胞菌（Pseudoalteromonas marina）鞭毛蛋白对生物被膜形成及厚壳贻贝附着的影响

大多数海洋无脊椎动物在发育过程中都经历浮游、底栖附着阶段,厚壳贻贝(Mytilus coruscus)作为海洋经济物种与大型污损生物,其附着机制受到广泛关注。为探究海洋细菌与厚壳贻贝附着的互作关系,选取了对厚壳贻贝稚贝附着具有较高诱导活性的海洋细菌—海假交替单胞菌(Pseudoalteromonas marina),采用酸解超速离心法提取P. marina的鞭毛蛋白。将提取的鞭毛蛋白与琼脂糖溶液混合,形成凝胶直接刺激稚贝;再用提取的鞭毛蛋白处理P. marina 生物被膜进行稚贝附着实验。通过共聚焦激光扫描分析形成的生物被膜上生物量、细菌密度和胞外产物含量的变化。结果表明:P. marina 鞭毛蛋白与琼脂糖形成的混合凝胶可显著促进厚壳贻贝稚贝的附着;鞭毛蛋白处理的生物被膜对厚壳贻贝稚贝附着的诱导活性显著提高;生物被膜上的生物量、细菌密度、膜厚、胞外β-多糖、脂质和蛋白浓度都有所增加。研究表明,鞭毛蛋白可以直接调控厚壳贻贝稚贝的附着,也可通过改变P. marina 生物被膜的生物学特性,间接影响厚壳贻贝稚贝的附着,为探究细菌鞭毛蛋白与厚壳贻贝附着互作机制提供理论依据。     

In vivo neutralization assays by monoclonal antibodies against white spot syndrome virus in crayfish(Cambarus proclarkii)

The neutralizing activities of eight monoclonal antibodies （MAbs） against white spot syndrome virus （WSSV） （2D2, 2B2,1D2, 1D5, 1C2, 4A1, 6A4 and 6B4） were analyzed by in vivo experiments. Gills from WSSV-infected shrimp were homogenized and ten-fold serially diluted by PBS, and then incubated with MAbs （hybridoma culture supernatant）, respectively. The mixture of WSSV and MAbs were injected into crayfish （Procambarus clarkii）. After challenge, the death rates of crayfish were counted to determine the neutralizing activities of MAbs. At the same time, the mixture of myeloma culture supernatant and WSSV or PBS was served as positive or negative control, respectively. The results showed that, at each virus dilution, the mean time to death of the crayfish injected with MAb-treated virus was significantly longer than that in the positive control, though they all showed 100% mortality within 25 d, and meanwhile, few crayfish died in the negative control. Among the eight MAbs, 2D2, 2B2, 1D2 and 1D5, especially the former two, delayed the mortality significantly, and 1 C2, 4A1 and 6A4 delayed the mortality as well but not so efficiently, while MAb 6IM was efficient only when the virus concentration increased. The results indicated that the anti-WSSV MAbs can neutralize WSSV in different virus dilutions.     

Production of an extracellular copper-binding compound by the heterotrophic marine bacterium Vibrio alginolyticus

The heterotrophic marine bacterium Vibrio alginolyticus was shown to produce extracellular copper-binding compound (s) when exposed to copper in a seawater medium. Fractionation and analysis of copper, and methionine incorporation in culture supernatant fractions showed that the copper-binding compound coeluted with material which was radiolabeled with ³⁵S methionine. This suggests that the copper-binding compound may be proteinaceous. Analysis of label incorporation from ¹⁴C glucose into the fraction containing the copper-binding compound indicated that it was produced actively by the cells during recovery from copper toxicity, and was not present because of non-specific release by lysed bacterial cells. Concentrated supernatants from control and copper-challenged cultures contained two compounds which could be marginally resolved by size exclusion HPLC (26 kD and 28 kD), and which were produced at about a ten-fold higher level in copper-challenged cultures than in controls. These data indicate that the bacteria respond to toxic copper levels by excretion of a ca. 28 kD compound (s), which serves to detoxify copper in the medium by formation of organic copper complexes.The data suggest a potential role for macromolecules excreted by heterotrophic bacteria in control of copper ion activity in seawater. The production of copper-complexing compounds in response to copper represents a mechanism through which bacteria may directly influence the speciation of metal ions in seawater.     

基于人为调控盐度的促进大叶藻种子萌发及建苗技术研究

Vast declines in Zostera marina seagrass beds demand effective methods of rehabilitation. In this study, we developed a practical method by reducing salinity to induce seed germination followed with recovering salinity to facilitate seedling production of Z. marina. The results showed that Z. marina seeds collected from natural seawater(salinity 30) were induced to germinate at reduced salinities. Percent germination(GR) was higher and mean-time-to-germinate(MTG) was shorter at lower salinities. The highest GR and shortest MTG occurred at salinity 0(deionized freshwater). After germination in freshwater, seeds could develop into seedlings at salinities5–30 and continue the growth. Viability or development of germinated seeds was not significantly different during the 40 d of post-germination incubation at salinities 5–15 after 1–20 d of germination in freshwater. However,during the process of translating germinated seeds from salinity 0 and 5 to salinity 30, reducing the gradients of post-germination acclimation facilitated more seeds forming seedlings in less time. On average, after 60 d of static incubation, including 20 d in freshwater for germination followed with immediate shift to salinity 5 and increasing to salinity 30 at increment of 5 every two days until cultivation at constant salinity 30, 33% of Z. marina seeds produced healthy seedlings. The results indicate that the salinity-manipulation based method of artificial germination and seedling production is practical and effective in supporting rehabilitation of Z. marina bed.     

天鹅湖大叶藻种苗补充情况调查

本研究以山东荣成典型潟湖——天鹅湖内的大叶藻(Zostera marina L.)海草床为研究对象,从2015年3月到6月对大叶藻种子萌发形成的幼苗进行进了月度调查,包括春季大叶藻种苗的密度、株高、叶鞘高、叶数、叶宽以及分枝情况。结果表明,天鹅湖大叶藻种子在3月即开始萌发,但萌发量较少,种苗密度较低;4月种子大量萌发,种苗密度不断升高,并达到峰值,为(481.77?303.42)株/m2;5月份种子萌发结束,种苗密度下降,同时开始克隆生长;6月份种苗不断生长,分枝数增多,种苗开始出现有性生殖枝。至此,春季种苗的萌发过程结束,进入有性生殖过程。     

河流弧菌(Vibrio fluvialis)对大黄鱼( Pseudosciaena crocea ) 鳃粘液粘附特性研究

采用间接ELISA法(最低检测值约104cfu)研究不同培养条件、不同培养阶段、抗体处理、营养饥饿等对河流弧菌粘附作用的影响。结果表明,经TSB培养的菌体的粘附作用极显著强于TSA培养的菌体的粘附作用(P<0.01);河流弧菌能很好地粘附于大黄鱼粘液,粘附量随菌浓度升高而增大并符合饱和粘附动力学:y=0.1782ln(x)1.6923(R2=0.9810);不同生长阶段河流弧菌的粘附能力不同,在培养初期阶段细菌的粘附量先是随着培养时间的延长而增大,并在培养24h后粘附量达到最大,而后随着培养时间的延长其粘附量急剧下降;抗体处理后河流弧菌的粘附量显著低于对照组(P<0.05);营养饥饿菌体的粘附量明显降低。以上结果表明:海水中的河流弧菌能很好地粘附于大黄鱼鳃粘液,其粘附作用受细菌培养条件、营养状况等自身因素的显著影响。本研究结果有助于了解河流弧菌的流行病学和致病机理。     

不同体系改性粘土对浒苔(Ulva prolifera)微观繁殖体去除及萌发的影响

本文研究了2种无机改性粘土在3种不同改性配比下对浒苔微观繁殖体去除效果及萌发的影响。结果发现,去除率随改性粘土及改性剂用量的增加而升高,相同条件下硫酸铝(AS)改性粘土对浒苔微观繁殖体的去除效果优于聚合氯化铝(PAC)改性粘土,其中1:5 PAC改性粘土在0.5 g/L时去除率为62%,AS改性粘土则可达到80%。添加改性粘土后上层水体中浒苔微观繁殖体相对萌发率随改性粘土及改性剂用量的升高而降低,总体系相对萌发率表现为先升后降趋势,在改性剂浓度为0.1 g/L时相对萌发率达到最高。两种改性体系中微观繁殖体在1:3配比、1.0 g/L浓度下均未能萌发。该结果显示,改性粘土可以有效去除浒苔微观繁殖体并抑制其萌发,这为我国绿潮灾害的防控提供了一种新思路。     

肠道细菌对厚壳贻贝稚贝附着的作用研究

为探讨肠道细菌形成的微生物被膜对贝类附着的作用,从厚壳贻贝(Mytilus coruscus)成体肠道中分离10株肠道细菌,调查厚壳贻贝肠道细菌形成微生物被膜特性,分析微生物被膜密度、细菌种属与厚壳贻贝稚贝附着之间的相互关系。所有肠道细菌均可有效促进稚贝附着。其中,Paracoccus sp.1微生物被膜诱导的稚贝附着率最高(61%),且微生物被膜膜厚较厚,细菌分布较密集;仅5株肠道细菌Roseovarius sp.1、Winogradskyella sp.1、Tenacibaculum sp.4、Bacillus sp.5和Nonlabens sp.1的被膜密度与附着显著相关(P0.05)。系统发育分析发现肠道细菌诱导附着率与其种属无关。本研究表明源于厚壳贻贝成贝肠道细菌可以促进其稚贝的附着,说明肠道细菌对厚壳贻贝的生长发育具有一定的作用。本研究结果为深入探讨肠道细菌与厚壳贻贝相互作用和推动该种规模化养殖提供理论依据。     

光棘球海胆(Strongylocentrotus nudus)酚氧化酶氧化产物的抗菌特性研究

本文通过线性连续梯度非变性电泳结合邻苯二酚发色法分离纯化了光棘球海胆(Strongylocentrotus nudus)体腔液上清中的酚氧化酶(PO),然后通过生长曲线测定法进行了光棘球海胆PO氧化产物的抗菌特性分析。结果显示:(1)光棘球海胆体腔液上清中存在3种PO(Sn PO1,Sn PO2,Sn PO3),在聚丙烯酰胺凝胶中与邻苯二酚反应分别呈现褐色、黄色和紫色;(2)以多巴胺为底物时,Sn PO1的氧化产物对灿烂弧菌Vibrio splendidus、哈维氏弧菌Vibrio harveyi和金黄色葡萄球菌Staphylococcus aureus有抗菌作用,Sn PO2氧化产物对哈维氏弧菌、金黄色葡萄球菌和拟诺卡式菌Nocardiopsis sp.有抗菌作用,Sn PO3氧化产物仅对哈维氏弧菌和拟诺卡式菌有抗菌作用;(3)以左旋多巴为底物时,仅Sn PO2氧化产物对拟诺卡式菌有抗菌作用;(4)光棘球海胆PO氧化产物对假交替单胞菌Pseudoalteromonas nigrifaciens、希瓦氏菌Shewanella baltica和溶壁微球菌Micrococcus lysodeikticus的生长无明显影响。上述结果表明,在光棘球海胆中,PO氧化产物具有窄谱抗菌活性,多巴胺氧化产物的抗菌活力高于左旋多巴氧化产物,并且不同PO亚型的氧化产物具有不同的抗菌谱和抗菌特性。     

拮抗菌对病原性溶藻弧菌(Vibrio alginolyticus)粘附大黄鱼(Pseudosciaena crocea)表皮粘液的影响

采用3H-TdR同位素示踪法研究了病原性溶藻弧菌和9株拮抗菌对大黄鱼表皮粘液的粘附动力学。结果表明,10株细菌的粘附作用都符合饱和粘附动力学特征;R4、R13和R32等3株拮抗菌的最大粘附量高于溶藻弧菌;9株拮抗菌的分离常数都高于溶藻弧菌;R32的亲和指数大于溶藻弧菌,其余8株拮抗菌的亲和指数都小于溶藻弧菌。测定了9株拮抗菌对病原性溶藻弧菌粘附大黄鱼表皮粘液的竞争、排斥和置换作用,结果显示:9株拮抗菌在竞争条件下都能极显著减少溶藻弧菌对表皮粘液粘附(P<0.01);在粘附排斥方面,R4、R13、J26、J28、J312、Y59等6株拮抗菌能显著排斥病原性溶藻弧菌对大黄鱼表皮粘液的粘附作用(P<0.05);在粘附置换方面,J26、J28等2株拮抗菌能极显著(P<0.01)降低病原性溶藻弧菌对大黄鱼表皮粘液的粘附量。研究结果表明,病原性溶藻弧菌对大黄鱼表皮粘液有较强的粘附作用;9株拮抗菌能够抑制溶藻弧菌对大黄鱼表皮粘液的粘附作用,其中竞争作用效果最好,排斥作用次之,置换作用的效果最差;拮抗菌对病原菌粘附的抑制效果受多种因素的影响,各菌株的粘附动力学参数有重要的影响作用。     

无机碳与雨生红球藻(Haematococcus pluvialis)细胞调节物质

以单细胞雨生红球藻为材料，采用酸碱滴定和CO2加富通气培养微藻的方法，对旧液中HCO3^-和CO3^2-浓度变化以及对红球藻细胞生长的影响进行了研究。结果表明，旧液具有限制红球藻细胞生长和诱导细胞转化的作用。同时，旧液中无机碳离子浓度明显高于新液。培养液中富含CO3^2-时，各细胞数量与CO3^2-浓度呈正相关，相关系数为0．88。溶液中仅有HCO3^-时，各细胞数与HCO3^-浓度也呈正相关性。因此，排除了CO3^2-和HCO3^-作为旧液中的调节物质，限制红球藻细胞生长和诱导细胞转化的可能性。旧液乙酸乙酯提取物生物检测实验表明，在粗提取物中有降低细胞增长和诱导细胞转化的活性，表明调节物质能溶于有机相，也反过来证实无机碳离子不是旧液中的调节物质。DNA含量和倍性分析结果表明，红球藻游动细胞DNA复制可以加倍后不经过原生质分裂就可以再次进行，因此推测旧液中的调节物质对原生质分裂过程产生抑制作用，而不对DNA复制过程产生抑制作用。     

海洋附着细菌对冠瘤海鞘幼体附着和变态的影响

从冠瘤海鞘、皱瘤海鞘及乳突皮海鞘的被囊表面及其附着基和附近的海水中分离到290株细菌,根据其形态和生理生化特征,将其鉴定至弧菌属、肠杆菌属和无色杆菌属等10个属.分析海鞘被囊表面及其附着基和海水中的细菌组成,并从各属中挑出的29株细菌中分离筛选出附着能力较强的9株.将这9株细菌分别制成不同的单种细菌黏膜及9种细菌混合黏膜,检测细菌黏膜对冠瘤海鞘幼体附着和变态的影响.结果表明不同细菌黏膜对冠瘤海鞘幼体的附着和变态起不同的作用.无色杆菌属H-13显著地促进海鞘的附着和变态;弧菌属的H-4对海鞘的附着和变态均起抑制作用;弧菌属H-9明显地促进附着却强烈抑制海鞘幼体变态;其余细菌仅仅对海鞘幼体附着或变态的一个过程起作用或是对两者都没有明显效果.     

改性粘土对浒苔(Ulva prolifera)微观繁殖体去除效果及萌发的影响

本文首先考察了不同浓度改性粘土对浒苔微观繁殖体的去除效果,实验结果显示,去除率随着改性粘土浓度增大而逐渐提高,0.4g/L的改性粘土对初始密度为4.8?105cell/m L的浒苔微观繁殖体去除率可达到80%以上。去除实验后继续研究微观繁殖体的萌发率发现,与对照组相比,不论是上层培养液中还是重新混合后的萌发率均随着改性粘土用量的增加而降低,1.0g/L的改性粘土可使其萌发率由60%左右降至低于6.5%。本文的研究结果表明,适量的改性粘土能有效去除海水中的浒苔微观繁殖体、降低其萌发率,有望成为消除或减弱绿潮灾害的有效手段。