凡纳滨对虾(Litopenaeus vannamei)造血激素的组织分布及细胞定位分析

造血激素(astakine)是一种具有促进造血组织细胞分化和增殖作用的甲壳动物细胞因子,参与甲壳动物的造血活动,对于只依赖于非特异性免疫的甲壳动物抗感染能力有重要的意义。本实验室前期成功克隆凡纳滨对虾造血激素(LvAST)的基因Lvast。为了进一步了解其功能,本文通过实时荧光定量PCR技术检测了Lvast表达的组织分布,同时还用Dlight标记抗LvAST抗体进行了LvAST在对虾细胞和组织中的免疫荧光定位分析。荧光定量PCR测定结果说明,Lvast主要在肝胰腺、鳃和肌肉中大量表达,而淋巴器官和血淋巴细胞中表达量较低;免疫荧光结果显示,LvAST可同时存在于血淋巴细胞内和血淋巴细胞膜表面,不同的血淋巴细胞表面的LvAST分布呈现出明显差异,可能具有重要的血细胞分类意义。LvAST蛋白在肝胰腺、鳃、肌肉、淋巴器官等组织中均有分布,并且肝胰腺组织中分布较多,淋巴器官组织中分布较少。     

凡纳滨对虾造血激素的组织分布及细胞定位分析

造血激素（astakine）是一种具有促进造血组织细胞分化和增殖作用的甲壳动物细胞因子,参与甲壳动物的造血活动,对于只依赖于非特异性免疫的甲壳动物抗感染能力有重要的意义。本实验室前期成功克隆凡纳滨对虾造血激素（LvAST）的基因Lvast。为了进一步了解其功能,本文通过实时荧光定量PCR技术检测了Lvast表达的组织分布,同时还用Dlight标记抗LvAST抗体进行了LvAST在对虾细胞和组织中的免疫荧光定位分析。荧光定量PCR测定结果说明,Lvast主要在肝胰腺、鳃和肌肉中大量表达,而淋巴器官和血淋巴细胞中表达量较低;免疫荧光结果显示,LvAST可同时存在于血淋巴细胞内和血淋巴细胞膜表面,不同的血淋巴细胞表面的LvAST分布呈现出明显差异,可能具有重要的血细胞分类意义。LvAST的蛋白在肝胰腺、鳃、肌肉、淋巴器官等组织中均有分布,并且肝胰腺组织中分布较多,淋巴器官组织中分布较少。     

一种甲壳动物血细胞分化因子性质的初步分析

血细胞是甲壳动物的免疫细胞,负责抵御和清除入侵的病原。虽然血细胞在甲壳动物的整个生命周期中都需要不断更新,但人们对甲壳动物造血机制的了解却十分有限。前期研究发现利用红螯螯虾的肌肉提取液可以诱导体外培养的造血组织细胞定向分化为颗粒细胞,但其中起到诱导分化作用的因子尚未明确。本研究对该未知血细胞分化因子的基本性质进行了分析。首先,我们制备了红螯螯虾（Cherax quadricarinatus）、中华绒螯蟹(Erinocheir sinensis)、东亚飞蝗(Locusta migratoria manilensis)、红石斑鱼(Epinephelus goreensis)、凡纳滨对虾(Litopenaeus vannamei)和疣荔枝螺(Reishia clavigera)的肌肉提取液,测试其诱导螯虾造血组织细胞分化的能力。结果表明,以上各种动物的肌肉提取液均可诱导原代培养的螯虾造血组织细胞向颗粒细胞分化,说明该血细胞分化因子普遍存在于这些动物的肌肉组织中。进一步我们以螯虾肌肉提取液为实验对象,分析了血细胞分化因子的热稳定性、溶解性、带电性、对蛋白酶的耐受性以及分子量大小。结果发现该血细胞分化因子易溶于甲醇,呈电中性,具有良好的热稳定性,且可耐受蛋白酶处理,分子量小于1 kDa。因此,螯虾肌肉提取液中可诱导造血组织细胞分化为颗粒细胞的血细胞分化因子是一种广泛存在的小分子类物质。本研究结果有助于后续对该因子的分离纯化和鉴定。     

适用于甲壳动物细胞的缓冲液配方优化

目前,在对螯虾、对虾等甲壳动物细胞的研究中,广泛采用了适用于哺乳动物细胞或昆虫细胞的各类缓冲液和培养液。但是,甲壳动物体液的渗透压与哺乳动物等有很大差异,直接沿用哺乳动物等细胞的缓冲系统不合适。本研究首先测定了红螯螯虾(Cherax quadricarinatus)、克氏原螯虾(Procambarus clarkii),与饲养在盐度30海水中的凡纳滨对虾(Litopenaeus vannamei)的血清渗透压,其值分别为(409±12)、(465±8)、(704±15) mOsmol/kg。随后,通过调节NaCl的浓度来改变抗凝剂和磷酸盐缓冲液(PBS)的渗透压,使其与相应动物的血清渗透压相等。优化后适用于红螯螯虾的抗凝剂和PBS中,NaCl的浓度分别为70、200 mmol/L;适用于克氏原螯虾的抗凝剂和PBS中,NaCl的浓度分别为100、230 mmol/L;适用于凡纳滨对虾的抗凝剂和PBS中,NaCl的浓度分别为220、360 mmol/L。进一步比较了3种动物的血细胞在不同渗透压缓冲液中的存活情况。结果表明,红螯螯虾、克氏原螯虾以及凡纳滨对虾这3种甲壳动物的血细胞在未优化的PBS中处理15 min,死亡率就分别达到4.5%、2.0%以及10.0%左右;然而当使用优化后的PBS进行处理时,死亡率则分别降至1.0%、1.0%以及2.0%左右。而血细胞在抗凝剂中的死亡率则较低(<2.0%),优化前后没有显著差异。综上,在甲壳动物活细胞实验中采用优化后的缓冲液,能改善细胞的状态,提高实验的准确性。     

红螯螯虾鳃细胞与血细胞分子标记的鉴定

甲壳动物的鳃中富集了大量血细胞,在循环血细胞稳态调节和免疫防御中发挥重要作用,因此获得区分鳃细胞和血细胞的分子标记将有助于研究鳃血细胞的功能。本研究采用SDS-PAGE电泳法比较了红螯螯虾(Cherax quadricarinatus)鳃细胞和血细胞的蛋白表达谱,获得3条组织特异性蛋白条带,并通过质谱技术鉴定了8个候选蛋白。实时荧光定量PCR(qRT-PCR)分析表明,空泡型-H⁺-ATP合酶C亚基(vacuolar-H⁺-ATPase subunit C, V-H⁺-ATPase)在鳃细胞中的转录水平是在血细胞中的53.3倍;而α-2-巨球蛋白(alpha-2-macroglobulin, α2M)和卵黄膜外层蛋白(vitelline membrane outer layer protein I-like protein, VMO-Ia)在血细胞中的转录水平分别是在鳃细胞中的10.3倍和10.9倍。Western-blot和免疫荧光分析表明,V-H⁺-ATPase和VMO-Ia蛋白分别在鳃细胞和血...     

甲壳动物化学感觉研究进展

本文从甲壳动物化学受体的形态、动物个体的行为反应以及化学感觉神经元的电生理三方面综述了甲壳动物化学感觉研究近20年的进展。水生动物,如甲壳动物特别是底栖甲壳动物,主要依赖化学感受器获得其生存环境的信息,从而表现出摄食、逃避捕食者、觅偶、交配、洄游等各种不同的行为反应。早期的研究主要是直观的行为观察和化学感受器的形态描述,内容偏重于动物的摄食习性和化学感受器的结构及其分布。随后电生理方法和电镜技术的引入,使化学感觉生理学的研究深入到细胞水平,发现了受体细胞的一些特性,如低自发放电频率、低阈值、调制、适应和去适应、范围分级等。另外,还提出了甲壳动物化学受体对化学信号的两种基本编码方式,即单线编码模式和交叉神经元模式。     

绿海龟(Chelonia mydas)血细胞发育过程的观察

采用Wright's液染色方法,对绿海龟的外周血、骨髓、脾脏和肝脏等几种组织的印片和涂片进行观察研究.结果表明,骨髓和脾脏是绿海龟的主要造血器官,红血细胞、粒细胞、单核细胞等血细胞主要在骨髓中发生,淋巴细胞主要在脾脏中发生;脾脏中可以发现晚幼红血细胞,脾脏可能是红血细胞的成熟场所;肝脏中无原始型的血细胞,可能不是其造血器官.红血细胞的发育经历了5个阶段:原红血细胞、早幼红血细胞、中幼红细胞、晚幼红血细胞和红血细胞等阶段,其胞体体积经历了由小到大,由大到小、再到大的发育过程;粒细胞的发育经历了5个阶段:原粒细胞、早幼粒细胞、中幼白细胞、晚幼白血细胞和白细胞等阶段,胞体体积均由大到小变化,核分叶可能是粒细胞的衰老的标志;淋巴细胞和单核细胞的发育各经历了3个阶段.各细胞发育均经历了由有细胞核仁到无细胞核仁的变化过程.原血细胞和幼血细胞在造血器官中可以进行有丝分裂,部分成熟的红血细胞可以在外周血中进行直接分裂.     

杂交石斑鱼Epinephelusfuscoguttatus♀×E.lanceolatus♂血细胞的类型及发生

为查明杂交石斑鱼Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂血细胞形态分类及血细胞发生特点,文章通过血液和组织涂(印)片、细胞染色和显微观察,研究了体重263.18±12.31g的杂交石斑鱼外周血液白细胞的分类组成,以及头肾、脾脏、体肾和肝脏等4种组织中各类血细胞的发生情况,并将结果与其母本进行比较。结果表明,杂交石斑鱼外周血液白细胞由淋巴细胞[(55.46±12.38)%]、血栓细胞[(21.11±5.38)%]、嗜中性粒细胞[(19.44±12.02)%]、单核细胞[(1.66±2.13)%]和嗜酸性粒细胞[(2.33±1.91)%]组成;4种组织印迹片中,头肾中未成熟的细胞总数比例最高[(16.12±0.30)%]],体肾次之[(7.90±0.35)%],脾脏[(2.56±0.21)%]和肝脏[(0.97±0.18)%]未成熟细胞比例较低;由此推断杂交石斑鱼主要造血组织是头肾,其次是体肾。在杂交石斑鱼4种组织中均观察到浆细胞,在头肾、体肾和脾脏印迹片中观察到嗜碱性粒细胞和巨噬细胞,在体肾印迹片中观察到巨大原单核细胞。杂交石斑鱼与其母本相比,外周血液白细胞中淋巴细胞比例接近(+2.16%),血栓细胞较少(-14.58%),嗜中性粒细胞(+9.10%)、嗜酸性粒细胞(+2.18%)和单核细胞(+1.38%)较多;主要造血组织都是头肾,但淋巴细胞的主要发生组织不同(分别是体肾和头肾),次要造血组织也不同(分别是体肾和脾脏);此外杂交石斑鱼各组织中未成熟细胞比例明显高于母本,显示出活跃的造血活动。本研究结果能为杂交石斑鱼的血液生理学、病理学研究及石斑鱼杂交选育提供基础资料。     

甲壳动物争斗行为研究进展

争斗残食已成为制约甲壳动物集约化养殖提质增效的瓶颈之一。甲壳动物的争斗行为受诸多外部因素影响,同时受机体生理代谢和基因调控。本文介绍了甲壳动物争斗行为的研究方法,概述了争斗行为的影响因素、生理代谢特征及基因调控等研究成果,提出了系统开展甲壳动物争斗行为量化的必要性及机制研究方向,以期为深入研究甲壳动物争斗行为奠定基础。     

中国对虾XO-SG复合体及其分泌物的初步研究

X器官-窦腺(XO-SG)复合体是甲壳动物重要神经内分泌器官，类似哺乳动物的下丘脑-垂体系统，它能分泌多种神经肽类激素，调控甲壳动物的生殖、发育和蜕皮等重要生理功能(Cooke et al.，1982； Keller，1992)。对XO-SG复合体的研究一直是甲壳动物内分泌学的重点内容。XO由视神经节内一群相互无连接的单轴突分泌肽神经细胞组成，它们的轴突末梢在血窦附近膨大形成窦腺，XO内胞体形成的神经肽类激素经轴突运输到窦腺储存和释放。近20年来，一些学者对10余种甲壳动物眼柄内X器官-窦腺复合体进了形态及超微结构研究，发现X器官神经内分泌细胞胞体的分布位置和结构特征各不相同，区分出2至7种不同类型的神经内分泌末梢，并观察到多种神经内分泌物质释放方式(Fingerman，1992； Dircksen，1992； Castany，1997； Weatherby，1981)。我国对甲壳动物神经内分泌系统的研究刚刚开始(蔡生力，1998)，仅对锯缘青蟹(Scylla serrata)、罗氏沼虹虾(Macrobrachium rosenbergii)和中华绒螯蟹(Eriocheir sinensis)XO-SG复合体进行了显微和超微结构观察(上官步敏等，1994，1995；姚泊，1995；孙金生等，2000)。中国对虾(Penaeus sinensis)是我国重要经济甲壳动物，在育苗过程中往往采用切断或灼烧眼柄的方法来加速亲虾性腺发育和产卵，而对XO-SG复合体却了解甚少，仅进行了组织学观察(康现江等，1998)。为此作者对中国对虾眼柄XO-SG复合体显微和超微结构及其分泌物特征进行了初步研究，为进一步研究对虾神经分泌细胞的离体培养和神经分泌调控机制打下基础。     

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.     

A Review of Some Environmental Issues Affecting Marine Mining

This article reviews information recently available from existing marine and coastal mining for responses to environmental issues affecting marine mining at different depths. It is particularly but not exclusively concerned with those issues affecting seabed biodiversity impact and recovery. Much information has been gathered in the past 10 years from shallow mining operations for construction aggregate, diamonds, and gold, from coastal mines discharging tailings to shallow and deep water, and from experimental deep mining tests. The responses to issues identified are summarized in a series of eight tables intended to facilitate site-specific consideration. Since impacts can spread widely in the surface mixing layer SML, and can affect the biologically productive euphotic zone, the main issues considered arise from the depth of mining relative to the SML of the sea. Where mining is below the SML, the issue is whether it is environmentally better to bring the extraction products to the surface vessel for processing (and waste discharge), or to process the extraction products as much as possible on the seabed. Responses to the issues need to be sitespecific, and dependent on adequate preoperational environmental impact and recovery prediction. For deep tailings disposal from a surface vessel, there are four important environmental unknowns: (1) the possible growth of "marine snow" (bacterial flocs) utilizing the enormous quantities of fine tailings particles (hundreds or thousands of metric tons per day) as nuclei for growth, (2) the possibility that local keystone plankton and nekton species may migrate diurnally down to and beyond the depth of deep discharge and hence be subjected to tailings impact at depth, (3) the burrow-up capability of deep benthos and their ability to survive high rates of tailings deposition, and (4) the pattern and rate of dispersion of a tailings density current through the deep water column from discharge point to seabed. Actions to obtain relevant information in general and site-specifically are suggested.     

Increased Particle Fluxes at the INDEX Site Attributable to Simulated Benthic Disturbance

Particle fluxes were measured 7 m above the sea bottom during the predisturbance, disturbance, and postdisturbance periods by using time series sediment traps attached to seven deep-sea moorings deployed in the INDEX experiment site in the Central Indian Basin. The predisturbance particle fluxes varied between 22.3 to 55.1 mg m -2 day -1 . Increased and variable particle fluxes were recorded by the sediment traps during the disturbance period. The increase observed was 0.5 to 4 times more than the background predisturbance fluxes. The increases in particle fluxes (~4 times) recorded by the sediment trap located in the southwestern direction (DMS-1) were the greatest, which could be the result of preferential movement of resuspended particles generated during the deep-sea benthic disturbance along the general current direction prevailing in this area during the experimental period. Also, the traps located closer to the disturbance area recorded greater fluxes than did the traps far away, across the Deep Sea Sediment Resuspension System path. This variability in recorded particle fluxes by the traps around the disturbance area clearly indicates that physical characteristics such as grain size and density of the resuspended particles produced during the disturbance had an important effect on particle movement. The postdisturbance measurements during ~5 days showed a reduction in particle fluxes of ~50%, indicating rapid particle settlement.     

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.     

Determination of In Situ Sediment Shear Strength from Advanced Piston Corer Pullout Forces

The advanced piston corer (APC) has been used by the Ocean Drilling Program since 1985 for recovering soft sediments from the ocean floor. The pullout force measured on extracting the core barrel from the sediment is shown to correlate with the average shear strength of the sediment core measured in the ship's laboratory. A simple rule of thumb is derived relating the shear strength of the sediment to the pullout force. Multiple APC holes at individual sites allow the consistency of the pullout measurements to be assessed. The effects of different operational procedures during APC coring are also explored. Although generally applicable, the correlation between pullout force and laboratory measurements of shear strength breaks down for some APC holes, possibly because of the disturbance of some sediment types during the APC coring process. A better understanding of the physical process of APC coring, and its effect on the properties of the sediment both inside and immediately outside the core barrel, would indicate what confidence can be put on the measurement of pullout force as a way of evaluating the in situ shear strength of deep sea sediments.     

《Acta Oceanologica Sinica》Vol. 33(2014) contents

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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,