深海贝莱斯芽孢杆菌DH82的抑菌活性物质初步分离纯化及其抑菌谱检测

贝莱斯芽孢杆菌(Bacillus velezensis) DH82发酵上清液经最佳硫酸铵饱和度(70%)沉淀、透析获得粗蛋白,粗蛋白经Sephadex G-75柱层析的色谱条件为柱1. 6 cm×50. 0 cm;上样量为5 mL;洗脱液为超纯水,流速2 mL/min;检测波长280 nm,获得两个具有抑菌活性的峰蛋白PrⅠ和PrⅡ。两个峰蛋白经抑菌谱检测,发现对常见水产指示病原细菌包括迟缓爱德华氏菌(Edwardsiella tarda)、嗜水气单胞菌(Aeromonas hydrophila)、坎氏弧菌(Vibrio campbellii)、溶藻弧菌(Vibrio alginolyticus)、哈维氏弧菌(Vibrio harveyi)、无乳链球菌(Streptococcus agalactiae)和金黄色葡萄球菌(Staphylococcus aureus)都具有较好抑菌活性,表明深海贝莱斯芽孢杆菌DH82产生的抑菌活性物质对水产病原细菌的抑菌谱较广,其中峰蛋白PrⅡ对指示植物病原真菌水贼镰刀菌(Fusarium equisetum)也具有较好抑菌作用。进一步对峰蛋白进行最小抑菌浓度(MIC)检验,发现其对迟缓爱德华氏菌和溶藻弧菌的MIC都较低(为7. 16μg/mL)。以上研究结果表明,深海贝莱斯芽孢杆菌DH82具有较宽的抑菌谱及较强的抑菌活性,预示该菌株在常见水产细菌性及个别植物真菌性病害防治上具有较好的开发利用价值。     

嗜水气单胞菌(Aeromonas hydrophila)引起斑马鱼肠道生理损伤和肠道菌群失调

研究分析了嗜水气单胞菌(Aeromonas hydrophila)感染斑马鱼后肠道生理健康和肠道微生物的变化。以斑马鱼为研究对象,刮伤皮下真皮,使用105 CFU/mL浓度的嗜水气单胞菌暴露6 h后转入清水,分别在暴露前、暴露后6 h、12 h、24 h取样。使用鱼特异性酶联免疫吸附试验(ELISA)试剂盒,检测肠道中紧密连接蛋白ZO-2 (TJP2)含量和乙氧基异戊二烯-O-脱乙基酶(EROD)酶活性,结果表明,嗜水气单胞菌暴露引起了斑马鱼肠道生理损伤,表现为肠道TJP2含量、EROD酶活性在暴露后显著下调。采用16S rRNA高通量测序检测肠道微生物及其菌群结构变化,嗜水气单胞菌暴露斑马鱼后致病菌不动杆菌属(Acinetobacter)明显增加,OTUs数量明显下降,Alpha多样性降低,结果说明嗜水气单胞菌引起了斑马鱼肠道菌群失调。肠道中微生物的多样性与改善肠道上皮和黏膜屏障功能紧密相关,该研究对从肠道健康的角度评价嗜水气单胞菌毒性,探索嗜水气单胞菌的致病机制具有重要意义。     

5 种抗生素对嗜水气单胞菌的抗菌后效应研究

采用菌落计数法测定了盐酸恩诺沙星、盐酸左氧氟沙星、氧氟沙星等5种抗生素对嗜水气单胞菌的抗菌后效应(PAE).结果显示,盐酸左氧氟沙星、氧氟沙星、盐酸沙拉沙星、盐酸恩诺沙星、烟酸诺氟沙星在1×MIC、2×MIC 和4×MIC 浓度时,对嗜水气单胞菌都有抗生素后效应,并随着抗生素浓度的增加,抗菌后效应的时间也延长.     

BSA-ISCOMs对欧洲鳗鲡免疫效应及抗细菌性病原的效果研究

为证实免疫刺激复合物(ISCOMs)的非特异免疫增强作用,将牛血清白蛋白(BSA)制备成ISCOMs(BSA-ISCOMs).将欧洲鳗鲡(Anguilla anguilla)随机分为3组,分别用BSA-ISCOMs、BSA和磷酸盐缓冲液(PBS,对照组)腹腔注射免疫.二次免疫完成后第30d采血,测血清部分非特异性免疫指标,剩余鳗鲡分别用创伤弧菌(Vibrio vulnificus)和嗜水气单胞菌(Aeromona hydrophila)攻毒.结果显示,BSA-ISCOMs组的血清溶菌酶活力显著高于BSA组和对照组(p≤0.05);超氧化物歧化酶(SOD)和补体C3活力与对照组无显著差异(p0.05).攻毒结果显示,BSA-ISCOMs组的存活率显著高于BSA组和对照组(p≤0.05),对创伤弧菌和嗜水气单胞菌攻击的相对免疫保护率分别为43.3%和51.7%.表明BSA-ISCOMs能有效增强欧洲鳗鲡对主要病原创伤弧菌和嗜水气单胞菌的抗病能力.     

ELISA技术在水产养殖病害诊断和海洋生物毒素检测中的应用进展

对ELISA技术原理及在水产养殖细菌性病害、病毒性病害及海洋生物毒素检测中的应用情况作一综述。ELSIA技术基础是抗原抗体特异性反应,样品与酶标记物和抗体在板孔中反应,洗去未结合的酶标记物,加入发色剂进行孵育,结合的酶标记物使发色剂颜色发生变化,最后加入终止液,在合适的波长下测定吸光度值。水产养殖中常见的细菌性病原为弧菌和嗜水气单胞菌,利用已建立的ELISA技术对大黄鱼肝脏中副溶血性弧菌含量进行测定,检出率为70%。嗜水气单胞菌的单克隆抗体技术分析也取得了较大进展,对于分泌抗嗜水气单胞菌单克隆抗体的细胞株,ELSIA分析可为快速、准确诊断鱼类嗜水气单胞菌提供可靠方法。20世纪90年代末,已有学者利用ELISA技术测定了草鱼出血病病毒(GCHV)的毒力,30 min内即可从未经提纯的毒液中检测出抗原,比较Dot-ELISA和SPA-CoA测定效果,发现灵敏度比SPA-CoA提高了10倍。对于虾类病毒性病原,ELISA测定主要涉及杆状病毒和白斑综合症病毒,其中对中国对虾杆状病毒检测灵敏度可达到60 ng,对同一个池白斑综合症发病率检测,检出率可达61.8%。利用ELSIA技术测定海洋生物毒素要求快速、方便、准确,日本于20世纪90年代初率先研制出检测DSP主要毒素软海绵酸(OA)的ELISA方法,美国和德国使用抗石房蛤毒素(saxtoxins,STX)和抗neo-STX多克隆抗体建立ELISA法测定PSP,可同时检测STX和neo-STX,灵敏度为40μg/(100 g)。虽然利用ELISA技术测定海洋生物毒素已经取得了一定进展,但是在检测过程中容易发生抗体交叉反应、系列标准毒素缺乏等因素仍然限制该方法的深入应用。     

斑节对虾病原菌胞外产物的致病性研究

由养殖濒死病虾体内分离的副溶血弧菌(2019#)、溶藻弧菌(4302#)和嗜水气单胞菌(1003#)培养液中分别提取了胞外产物,测定了酶成分,定量分析了蛋白酶及磷酸酯酶的活性.用细菌胞外产物粗提液对斑节对虾进行致死试验,戊二醛固定致死对虾的肝组织,电镜观察了斑节对虾肝组织的超微病变,初步探讨了三株细菌胞外产物诱发虾病的病理.     

中国对虾血淋巴抗菌活力的测定

采用溶藻胶弧菌（Ｖｉｂｒｉｏ ａｌｇｉｎｏｌｙｔｉｃｕｓ）、副溶血弧菌（Ｖ． ｐａｒａｈａｅｍ ｏｌｙｔｉｃｕｓ）、哈维氏弧菌（Ｖ．ｈａｒｖｅｙｉ）、嗜水气单胞菌（Ａｅｒｏｍ ｏｎａｓｈｙｄｒｏｐｈｉｌａ）对中国对虾（Ｐｅｎａｅｕｓ ｃｈｉｎｅｎｓｉｓ）血浆样品、血细胞溶解物（ＨＬＳ）样品的抗菌活力进行了测定。表明中国对虾血浆样品对四种细菌均表现出一定的抗菌活力。其中,三种弧菌活细胞数在孵育１ｈ 时呈现最低;嗜水气单胞菌活细胞数在孵育４ｈ 时最少。在三种弧菌中,血浆样品对哈维氏弧菌表现出的抗菌活力最强,而对副溶血弧菌表现出的抗菌活力最弱。血细胞溶解物样品对三种弧菌几乎没有表现出抗菌活力;嗜水气单胞菌在与血细胞溶解物样品孵育２ｈ 时,其活细胞数呈现最低,说明血细胞溶解物样品对该菌表现出了一定的抗菌活力。由此显示,抗菌活力在中国对虾血浆样品中表现得较强,而在血细胞溶解物样品中表现得较弱,血淋巴抗菌因子可以在短时间内对细菌表现出其最强的活力,但对不同种细菌其抗菌作用强弱不同。     

苯并异噻唑啉酮类衍生物的制备与生物活性测试

以1,2-苯并异噻唑-2(3H)-酮为原料合成了3-羰基苯并异噻唑-2(3H)-苯甲酸甲酯衍生物,目标化合物的结构经元素分析、<'1>H NMR和质谱确证.化合物对大肠杆菌、金黄色葡萄球菌、溶藻弧菌、嗜水气单胞菌、枯草芽孢杆菌5种海洋菌的抑菌活性结果表明,化合物对上述菌种均有不同程度的抑制作用,其中对大肠杆菌活性最高,...     

3种水产病原菌简型基因芯片检测技术的建立

根据水产养殖中常见的3种病原菌鳗弧菌(Vibrio anguillarum)、嗜水气单胞菌(Aeromonas hydrophila)及迟缓爱德华氏菌(Edwardsiella tarda)的研究资料,筛选3个毒力相关基因toxR、aerA、evpA设计引物和探针,构建简型基因芯片,并使用对虾白斑病综合征病毒(WSSV)variable region的PCR荧光标记产物作为表面化学质控。通过已构建和优化的多重PCR反应条件,获得了3个目的基因的PCR产物;经过芯片制作过程的优化,将探针以终浓度20μmol/L溶于50%DMSO,在室温、相对湿度45%的条件下点印于醛基基片表面。扩增的PCR产物与杂交液混合后,在42℃杂交2 h就可检测到理想的杂交信号。芯片的灵敏度试验结果表明,可以检测到的3种水产病原菌的最低模板DNA为:鳗弧菌3×102拷贝、嗜水气单胞菌5×103拷贝、迟缓爱德华氏菌6×101拷贝。该芯片可成功应用于患病大菱鲆内脏的细菌分离物的鉴定。     

基于核酸适配体的差减荧光法检测鳗弧菌(Vibrio anguillarum)

鳗弧菌(Vibrio anguillarum)可感染鳗鲡、虹鳟和大菱鲆等多种水产动物,是水产养殖中的一种重要病原菌,对其进行快速检测是病害防控的前提和基础。利用鳗弧菌与其核酸适配体之间有较强的亲和特异性,首次建立了一种基于核酸适配体的可定量检测鳗弧菌的差减荧光法。该方法对鳗弧菌有较好的特异性,对鳗弧菌的检测荧光值是其他菌(溶藻弧菌、哈维氏弧菌、铜绿假单胞菌、变形假单胞菌、嗜水气单胞菌、迟钝爱德华氏菌和大肠杆菌)的4～11倍,对鳗弧菌的最低检测限为10²CFU/mL,可用于10²～10⁸CFU/mL的范围内的定量检测。通过对不同盐度海水和鱼体组织样品进行加标回收检测,结果表明,回收率和相对标准偏差等指标均符合相应的标准,说明该检测方法可用于海水样品和水产动物组织中鳗弧菌的检测。     

Microbial Biomass and Organic Nutrients in the Deep-Sea Sediments of the Central Indian Ocean Basin

In order to assess the impact of deep-sea mining on the in situ benthic life, we measured the microbial standing stock and concentration of organic nutrients in the deep-sea sediments of the Central Indian Ocean Basin in the Indian pioneer area. Sediments were collected using box core and grab samples during September 1996. The total bacterial numbers ranged from 10 10 -10 11 cells per g -1 dry weight sediment. There was a marginal decrease in the number of bacteria from surface to 30 cm depth, though the subsurface section registered a higher number than did the surface. The highest numbers were encountered at depths of 4-8 cm. The retrievable number of bacteria were two orders less in comparison with the direct total counts of bacteria. An almost homogeneous distribution of bacteria, total organic carbon, living biomass, and lipids throughout the depth of cores indicates active microbial and benthic processes in the deep sea sediments. On the other hand, a uniform distribution of total counts of bacteria, carbohydrates, and total organic carbon in all the cores indicates their stable nature and suggests that they can serve as useful parameters for long-term monitoring of the area after the benthic disturbance. Further studies on temporal variability in this region would not only verify the observed norms of distribution of these variables but would also help to understand restabilization processes after the simulated benthic disturbance.     

Assessing the Validity of Seismo-Acoustic Predictor Equations for Obtaining Seabed and Sub-Surface Sediment Physical Properties

The interdependence between the seismo-acoustic properties of a marine sediment and its geotechnical/physical parameters has been known for many years, and it has been postulated that this should allow the extraction of geotechnical information from seismic data. Though in the literature many correlations have been published for the surficial layer, there is a lack of information for greater sediment depths. In this article, a desktop study on a synthetic seafloor model illustrates how the application of published near-surface prediction equations to subsurface sediments (up to several tens of meters burial depth) can lead to spurious predictions. To test this further, acoustic and geotechnical properties were measured on a number of sediment core samples, some of which were subjected to loading in acoustically-equipped consolidation cells (oedometers) to simulate greater burial depth conditions. For low effective pressures (representing small burial depths extending to around 10 meters subsurface), the general applicability of established relationships was confirmed: the prediction of porosity, bulk density, and mean grain size from acoustic velocity and impedance appears generally possible for the investigated sedimentary environments. As effective pressure increases through, the observed relationships deviate more and more from the established ones for the near-surface area. For the samples tested in this study, in some instances increasing pressure even resulted in decreasing velocities. There are several possible explanations for this abnormal behavior, including the presence of gas, overconsolidation, or bimodal grain size distribution. The results indicate that an appropriate depth correction must be introduced into the published prediction equations in order to obtain reliable estimates of physical sediment properties for greater subsurface depths.     

海上大直径钢管桩打桩过程中桩周土强度弱化研究

海上大直径钢管桩打桩过程中,桩周土体受到强烈扰动而发生强度弱化,掌握桩周土体强度弱化规律对于准确预测打桩过程、保证工程安全具有重要意义。为研究土体强度弱化规律,开展了环剪试验模拟打桩对桩周土体的扰动,测试土体强度随剪切速率的变化规律,建立了描述土体强度弱化规律的拟合公式,引入到打桩分析软件中。研究结果表明：土体的强度折减程度不仅与土体本身的性质有关还受到土体的埋深和剪切速率的影响,埋深越深土体强度折减程度越低,剪切速率越高土体强度折减越高,在打桩分析中可采用这里推荐的线性折减方法来模拟不同深度处土体强度的折减规律。     

Geoacoustic Inversion via Genetic Algorithm and Its Application to Manganese Sediment Identification

An acoustic inversion method using a wide-band signal and two near field receivers is proposed and applied to multiple layered seabed models including a manganese sediment. The inversion problem can be formulated into a probabilistic model comprised of signals, a forward model, and additive noise. The forward model simulates wide-band signals, such as chirp signals, and is chosen to be the source-waveletconvolution plane wave modeling method. The wavelet matching technique, using weighted least-squares fitting, estimates the sediment sound-speed and thickness on which determination of the possible numerical ranges for a priori uniform distribution is based. The genetic algorithm is applied to a global optimization problem to find a maximum a posteriori solution for determined a priori search space. Here the object function is defined by an L 2 norm of the difference between measured and modeled signals. Not only the marginal pdf but also its statistics are calculated by numerical evaluation of integrals using the samples selected during importance sampling process of the genetic algorithm.     

Interannual Sea Level Variations and Annual Tides in the Northwestern Pacific

Specific properties of the interannual sea level variations and annual tides in the Northwestern Pacific were studied. Several tide stations were monitored. The monthly mean sea level for the year of 1995 was analyzed at each tide station. A seismic event in 1995, some tectonic activity around the subject area, and the Kuroshio (the oceanic western boundary current) may possibly contaminate results which would have occurred from the astronomical annual tide alone.     

Flow Cytometric Analysis of Coastal Lagoon Bacterioplankton and Picophytoplankton: Fixation and Storage Effects

The effect of different fixation and storage protocols on the flow cytometric (FCM) simultaneous analysis of bacterioplankton and phytoplankton in coastal seawater samples (Mediterranean coastal lagoons) was investigated. FCM measurements (cell number, fluorescence and scatter characteristics) were obtained through DAPI staining. Three fixatives [glutaraldehyde (GA), formaldehyde (FA) and paraformaldehyde (PFA)] and two storage (3 months duration) methods (5 °C and −196 °C) were tested. Two dominant populations were detected in studied samples: bacteria and eukaryotic picophytoplankton. Adding fixatives (2% final concentration) appears necessary to obtain FCM exhaustive counts of all the bacteria and phytoplanktonic cells. This was related to the permeation effect of fixatives which allowed a better DAPI staining of the cells. Maximum fluorescence, i.e. optimal staining of the cells was obtained with FA or PFA, and significant lower fluorescences with GA. Fixed samples stored at 5 °C induced rapid cell loss. Only storage in liquid nitrogen of samples fixed with FA or PFA, allows mid-term (≥4 months) preservation of bacteria or picophytoplankton cell numbers, and limited evolution of DAPI-induced fluorescence and scatter characteristics.     

INFORMATION FOR AUTHORS

<正>Acta Oceanologica Sinica(AOS)is a comprehensive academic journal edited by the Editorial Committee of Acta Oceanologica Sinica and is designed to provide a forum for important research papers of the marine scientific community which reflect the information on a worldwide basis.The journal publishes scholarly papers on marine science and technology,including physics,chemistry,biology,     

Deep-Sea Sediment Compression Curves: Some Controlling Factors, Spurious Overconsolidation, Predictions, and Geophysical Reproduction

Oedometer tests have been carried out on 70 undisturbed surficial clays (at approximately 250 mm below the mudline), mostly collected by free-fall corers from sites widely scattered throughout the deep-sea North Atlantic. Acoustic measurements were also made, initially on contiguous samples and ultimately on the same sample using a geophysically instrumented oedometer which also collected electrical resistivity data. Apart from those quiescent areas below the carbonate compensation depth, such as north of the West Indies where very fine clays exist, most of the samples are silty clays whose geotechnical-geophysical properties are dependent on the type of clay minerals present (and their ability to take in moisture), the sand-size fraction, and the quantity of carbonate present. Thus the pure clays have high compressibilities which decrease on the addition of coarse particles, while the converse is true for the acoustic parameters, these increasing with the sand fraction. Using the notion of the intrinsic compression line for all samples, and comparison to it of the measured compression curves, it is clear that, contrary to some previously held ideas, most deep-sea clays are normally consolidated; the addition of carbonate has the effect of creating an open, stronger sediment skeleton. Interestingly, where information is available, the variation with depth of a sample's acoustic velocity follows the void ratio pressure relationship of the compression curve. This allows the construction of an in-situ sediment compression curve using the in-situ geophysical observations.