基于RNA-seq数据分析长牡蛎(Crassostrea gigas)天然免疫的可变剪接事件

大部分无脊椎动物缺乏基于经典抗体和记忆细胞的适应性免疫, 因而天然免疫在无脊椎动物免疫防御中发挥不可或缺的作用。可变剪接正是产生天然免疫多样性和特异性的重要途径。牡蛎(Crassostrea)是全球范围内的大宗养殖贝类和我国产量最高的海水养殖贝类, 理解其天然免疫系统的多样性和特异性对牡蛎病害防治和养殖业健康可持续发展至关重要。通过对不同病原诱导以及诱导后不同时间的转录组进行生物信息分析, 系统地研究了长牡蛎天然免疫应激反应的可变剪接事件。发现在弧菌诱导下可变剪接事件的总数显著增加, 表明弧菌的感染会诱导长牡蛎可变剪接事件的产生。对病原诱导后可变剪接体的组成类型及表达量显著变化的基因的功能富集分析, 表明免疫系统相关功能被显著富集, 病原感染不同阶段以及不同病原感染后可变剪接体的组成类型和表达量均不一致, 表明长牡蛎免疫系统可通过可变剪接产生免疫响应的特异性和多样性。研究结果为长牡蛎等无脊椎动物天然免疫多样性和特异性提供了典型实例, 也为长牡蛎病害防御提供了理论支撑。     

盐度对近江牡蛎幼虫生长及存活的影响

采用室内培育手段,研究了盐度对近江牡蛎(Crassostrea rivularis)幼虫生长及存活的影响。将人工授精孵育的面盘幼虫分别置于盐度依次为16,25,30的砂滤海水中培育至眼点幼虫。每隔一天检测各盐度组幼虫的壳长、壳高和存活数量。结果表明,16盐度组的幼虫生长最快,25盐度组次之,30盐度组最慢。从受精后第14天开始,16盐度组幼虫的生长显著快于30盐度组(P<0.05)。在整个浮游幼虫阶段,高盐度组幼虫的存活率低于低盐度组,其中30盐度组的存活率最低。16和25盐度组在幼虫出现眼点之前均出现大量死亡现象,而30盐度组并没有出现大量死亡现象。上述结果说明,近江牡蛎幼虫经过高盐驯化,对高盐环境的抗性具有一定的提高。     

长牡蛎可溶性黑色素的脱盐研究

该研究是继天然长牡蛎(Crassostrea gigas)黑色素的提取、可溶性长牡蛎(Crassostrea gigas)黑色素的制备之后,对牡蛎黑色素提纯方面的进一步拓展和延伸。运用半透化学材料分离的方法,选取纳滤透析膜为分离介质,对可溶性长牡蛎黑色素的制取产物中多余的盐类无机物进行分离剔除,进而得到纯度较高、品质优良的可溶性长牡蛎黑色素晶体,通过称质量和盐度测量,计算得出该方法对黑色素脱盐率达到98%,证明了该方法可以进行贝类黑色素的脱盐处理工作。     

珠母贝亲贝催熟培育和催产技术的研究

对不同来源珠母贝(Pinctada margaritifera)亲贝分别进行海上浅吊疏养促熟和室外水泥池人工催熟培育,且采用阴干、藻液、温度、低盐度刺激和异性产物诱导亲贝排放精卵,比较不同方法、不同来源亲贝性腺发育及亲贝开始排放时间、排放率、受精率和幼虫畸形率情况.结果表明,室外水泥池人工催熟法亲贝性腺成熟度比浅吊疏养促熟法好、死亡率低;相同培育方法,北部湾亲贝比海南亲贝性腺成熟快,但死亡率高;阴干刺激3 h+藻液刺激1 h+异性产物诱导,性腺达到第四期(排放期)的亲贝才排放,且排放的性细胞成熟度好,受精率高,幼虫畸形率低.     

The Role of Suspended Oyster Culture on Nitrogen Cycle in Hiroshima Bay

The predominance of bivalves affects the cycle of materials in the coastal ecosystem. In the present study, the role of suspended oyster culture on the nitrogen cycle was demonstrated for the northern Hiroshima Bay. The nitrogen cycle was considered as two systems, (1) the primary production (PP) system and (2) the oyster culture (OC) system. The results show that about 26% of N productivity was supplied to process by cultured oysters in the OC system. This process varies seasonally due to the seasonal variations of PON, physiological activities and biomass of oysters. The N processing rates were found to be high in summer and low in winter. The biodeposition and excretion of N in the OC system are 3.0 and 2.1 ton N d^–1, while the natural sedimentation rate and N regeneration in the PP system are 8.3 and 18.0 ton N d^–1, which indicates that the PP system is a major system regenerating N in the water column. The release of total dissolved N from the bottom to the water column is about 8.3 ton N d^–1. The amount of N harvested as oyster product was about 1.3 ton N d^–1, which is about 10% of daily N loading in north Hiroshima Bay. According to the N cycle developed in the present study, the results suggest the significant role of suspended oyster culture on the nitrogen cycle in Hiroshima Bay. In addition, our results indicate that oyster production was efficiently harvested, suggesting that oyster culture could probably be used as a tool to remove N from Hiroshima Bay.     

两个地理群体香港牡蛎(Crassostrea hongkongensis)杂交子代早期生长发育的杂种优势研究

为探讨不同地理群体香港牡蛎的杂种优势,于2014年5月开展了广东珠海(Z,生长快)和广西茅尾海(M,抗逆性强)群体间的2×2双列杂交,比较了各实验组早期生长、存活优势,并计算了各阶段的杂种优势,实验由2个杂交组MZ(M♀×Z♂)、ZM(Z♀×M♂)和2个自交组MM(M♀×M♂)、ZZ(Z♀×Z♂)组成。结果表明:浮游期间,正交组(MZ)幼虫在9日龄前未表现出生长优势,但表现出一定的存活优势,幼虫大小存在显著的母本效应;12日龄时正交杂种优势5.34。反交组(ZM)幼虫生长和存活都表现出了正杂种优势,但生长优势值逐渐减小,到了12日龄时壳高与自繁组(ZZ)幼虫间差异不显著(P0.05)。室内苗种培育阶段,杂交稚贝表现出明显的生长、存活优势,正交组(MZ)和反交组(ZM)子代的生长优势分别为7.86±3.12、17.47±13.14;存活优势分别为8.33±1.41、2.86±1.08。以上结果说明两个群体香港牡蛎杂交可以产生杂种优势,可为牡蛎杂种优势开发利用提供参考。     

Phylogenetic,histological and age determination for investigation of non-native tropical black-lip pearl oyster, <Emphasis Type="Italic">Pinctada margaritifera</Emphasis>, settled in jeju,Korea

This study reports the first finding of tropical blacklip pearl (BLP) oyster, Pinctada margaritifera, in Korean waters. One BLP oyster was found and sampled from Beom-seom islet (33°13′N, 126°33′E) in the southern coast of Jeju Island, Korea in February, 2011. The taxonomic status of the specimen was confirmed by nucleotide sequences of the mitochondrial gene cytochrome oxidase I and 28S ribosomal DNA. Oxygen isotopic analysis (δ¹⁸O_sh) and histology were used to determine the specimen’s age and reproductive stage, which were 3.5 years and ripe male, respectively. This BLP oyster most likely originated in an area in the subtropics or tropics, such as the Okinawa archipelagic or Micronesian regions. We suggest that intensive monitoring surveys are necessary for confirming the northward expansion of BLP oyster in Korean waters in the near future.     

Zinc concentration effect at the organismal,cellular and subcellular levels in the eastern oyster

The purpose of this research was to demonstrate a concentration effect of zinc exposure at organismal, cellular and sub-cellular levels in the eastern oyster and to find associated protein expression signatures (PES) for each concentration of zinc. Oysters were exposed to six concentrations of zinc for 48 h in a controlled environment. At the organismal level, fecal material was observed as a measure of physiological health during metal exposures. At the cellular level, lysosomal destabilization was measured using hemolymph. This cellular response was significant only at the highest concentration, when the fecal index was lowest. Protein responses were monitored in the oyster following exposure to zinc. Gill tissue was excised and homogenized, and then analyzed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and digital image analysis. Protein expression signatures were found to be specific to each concentration. The protein responses were linked to the other biological parameters measured, each of which followed a concentration gradient of zinc.     

双酚A暴露对不同黑色素含量长牡蛎的影响研究

长牡蛎的壳色有很多种,其中壳色较深个体(黑壳)比较浅个体(白壳)的外套膜中含有更多的黑色素。本研究采用高效液相色谱-质谱法(HPLC-MS/MS)测定天然海水中白壳与黑壳长牡蛎外套膜双酚A (BPA)含量,发现黑壳长牡蛎外套膜中的BPA含量较白壳长牡蛎低(P0.05)。由于黑色素合成过程中的关键——酶酪氨酸酶是一种酚氧化酶,可以通过对氧化酚类或芳胺类等多种底物的氧化起催化作用,推测其在BPA等酚类毒物的降解中也发挥重要作用。为了进一步研究不同黑色素含量牡蛎对BPA降解能力和对BPA损伤抵抗能力的差异,本实验进行了浓度为1 mg/L,1.5 mg/L和2 mg/L的BPA亚慢性暴露实验:通过对暴露后长牡蛎外套膜组织中BPA含量分析发现,白壳与黑壳长牡蛎外套膜中BPA含量均随着暴露浓度的升高而升高,且3种浓度BPA亚慢性暴露后,黑壳长牡蛎外套膜BPA含量均低于白壳长牡蛎,但是差异不显著(P0.05)。采用3种浓度BPA亚慢性暴露后,黑壳长牡蛎外套膜组织中活性氧(ROS)荧光值均显著低于白壳长牡蛎(P0.01)。通过对外套膜组织学观察发现,2 mg/L的高浓度BPA暴露使白壳长牡蛎外套膜组织发生了明显的病理变化,黑壳长牡蛎外套膜未发生明显的病理变化。可见,黑壳长牡蛎对BPA暴露可能具有更强的抵抗能力。     

乳山长牡蛎(Crassostrea gigas)的抗性基因表达和生存环境的季节差异

长牡蛎 （Crassostrea gigas） 是我国重要的海水养殖贝类,近年其大规模死亡现象频繁发生。为了解长牡蛎的免疫基因在季节上的表达模式变化规律及死亡爆发高峰期的水环境情况,在其主养区山东乳山进行了相关研究。分别在 2018 年 1、 3、5—9 月对该地区牡蛎鳃样品做了免疫基因定量分析,在 6、7、9 月对养殖海域的环境因子 （水温、盐度、pH、溶解氧）、各时间点水体里的浮游植物数量及各采样点牡蛎条件指数的变化进行了研究。结果显示,HSP70 在 7 月份高表达,其余 5 个抗性相关基因在 8 月表达水平达到峰值。在测量的三个月中,7 月水温最高,6 月和 9 月水温略低于 7 月,这一结果和表达模式相关。研究表明近岸的藻类丰度更大。因此,温度是影响长牡蛎存活的各环境因子中的主要因素,温度显著影响到体内抗性基因的表达情况并且间接影响到夏季牡蛎的条件指数。     

Distribution and Diversity of Ibla cumingi Darwin [Crustacea, Cirripedia) from the Gulf of Elat (Red Sea)

Abstract. The distribution of Ibla cumingi DARWIN on different types of hard substrata along 30 km of coast in the Gulf of Elat was studied. I. cumingi was found among and underneath Tetraclita squamosa rufotincta , in oyster beds, underneath slabs of beachrock, and in fissures in igneous rocks. In the Tetraclita belt, I. cumingi is most frequent in empty shells of dead Tetraclita while in the oyster bed it is common inside the meshwork made by the oysters. The distribution is regarded as clustered distribution. The density and size of the clusters of I. cumingi is determined by the available sheltered space within the midlittoral zone. The form of the habitat determines the shape of the animals. Those found in the oyster bed and fissures are usually longer than those found in the Tetraclita belt due to the depth of the fissures or to the bigger spaces within the oyster bed. I. cumingi is orientated so that the capitulum with the cirri is projecting towards the open water, and the water current. It is concluded that I. cumingi can withstand the physical stress encountered in a tropical intertidal zone, but that the distribution pattern is caused by predation.     

褶牡蛎垦区垂下式养殖的探讨

褶牡蛎(Ostrea plicatula Gmelin)是我省四大养殖贝类之一,它具有资源丰富、适应性强、分布广、养殖周期短等特点,其味道鲜美、营养丰富并有药用价值,是出口的水产品之一。 褶牡蛎的养殖有悠久的历史,但历年来只停留在潮间带养殖。由于潮间带生长的牡蛎摄食时间短,敌害多,所以产量低、个体小,每亩(1000块条石)年产蛎肉250公斤左右,并且养殖业的发展也受海区条件的限制。为了提高牡蛎的产量及垦区的利用率,1979年开始在莆田县忠门乌垞大队综合场进行褶牡蛎垦区垂下     

长牡蛎Wnt5b基因的鉴定及在黑色素生成中的作用

Wnt基因家族编码一类保守的分泌型糖蛋白,广泛参与细胞分化与增殖、细胞运动、凋亡、免疫调控以及色素生成等生物学过程。本研究鉴定了一个长牡蛎Wnt家族基因——CgWnt5b,比较了黑、白壳色长牡蛎中CgWnt5b转录水平的表达情况,并对其在调控黑色素生成方面的功能进行初步研究。研究结果显示,CgWnt5b编码序列全长1116 bp,编码371个氨基酸。生物信息学分析预测CgWnt5b蛋白相对分子量为42 KDa,等电点为9.28。对CgWnt5b与哺乳动物Wnt基因家族19个成员进行系统发育分析,确定了CgWnt5b为哺乳动物Wnt5a和Wnt5b的同源基因。荧光实时定量PCR检测长牡蛎外套膜、鳃、消化腺、闭壳肌、性腺、唇瓣以及血淋巴组织中CgWnt5b的mRNA分布,结果显示,CgWnt5b在外套膜、闭壳肌和唇瓣组织中的相对表达水平较高,在消化腺、鳃、血淋巴和性腺组织中相对表达量较低。黑壳长牡蛎中CgWnt5b mRNA表达量显著低于白壳长牡蛎,且siRNA敲降CgWnt5b的表达后,多个黑色素生成相关酪氨酸酶基因的表达显著上调。综上,本研究在长牡蛎中鉴定了一个Wnt家族成员CgWnt5b,可能通过抑制黑色素生成关键基因TYR的表达抑制长牡蛎中黑色素的生成。     

Cloning a new cytochrome P450 isoform (CYP356A1) from oyster Crassostrea gigas

We have cloned the full-length cDNA of the first member of a new cytochrome P450 (CYP) family from the Pacific oyster Crassostrea gigas. This new CYP gene was obtained based on an initial 331bp fragment previously identified among the list of the differentially expressed genes in oysters exposed to untreated domestic sewage. The full-length CYP has an open reading frame of 1500bp and based on its deduced amino acid sequence was classified as a member of a new subfamily, CYP356A1. A phylogenetic analysis showed that CYP356A1 is closely related to members of the CYP17 and CYP1 subfamilies. Semi-quantitative RT-PCR was performed to analyze the CYP356A1 expression in different tissues of the oyster (digestive gland, gill, mantle and adductor muscle). Results showed slightly higher CYP356A1 expression in digestive gland and mantle, than the other tissues, indicating a possible role of the CYP356A1 in xenobiotic biotransformation and/or steroid metabolism.     

牡蛎幼虫光照实验提示复杂遗传背景下基因互作的多样性

为研究光照对牡蛎幼虫生长成活率的影响,使用6组牡蛎幼虫家系,前三组为长牡蛎家系(GG1、GG2、GG3),后三组为长牡蛎与葡萄牙牡蛎杂交家系(GA1、GA2、GA3),其中GG1与GA1母本相同,GG2与GA2母本相同,GG3与GA3母本相同;每个家系分别在6个光照梯度下从1日龄饲养到10日龄,然后测量记录10日龄幼虫在各个光照梯度下的壳高和成活率。结果表明,在各个光照梯度下,牡蛎幼虫的壳高都没有显著差异,长牡蛎幼虫与杂交牡蛎幼虫之间的壳高也都没有显著差异,但长牡蛎幼虫和杂交牡蛎幼虫均在58 lx的光照强度下,表现出最高的成活率,这提示牡蛎育苗生产的前10天应当保持这样的光照强度,而杂交牡蛎幼虫在269 lx和58 lx两个光照梯度下的成活率均极显著的高于长牡蛎幼虫,提示了长牡蛎与葡萄牙牡蛎之间具有亲和性并且其杂交后代存在杂种优势,也在一定程度上支持长牡蛎与葡萄牙牡蛎同属一个物种的假设。而以壳高和成活率作为指标进行聚类分析和主成分分析后发现,长牡蛎3个家系幼虫的表型比较接近,而杂交牡蛎幼虫的表型虽然具有靠近各自同母本长牡蛎幼虫的趋势,但杂交家系之间差异非常大,提示了高杂合度高异质性遗传背景下基因互作的多样性。     

酸碱粗提配合超声破碎法制取可溶性长牡蛎(Crassostrea gigas)黑色素的研究

虽然牡蛎黑色素已经提取鉴定成功,但因为大部分实验需要用到黑色素的水溶液,而天然黑色素不溶于水,致使天然长牡蛎(Crassostrea gigas)黑色素的功效验证工作难以开展。本课题组探究总结出一套有效方法,对可溶性长牡蛎黑色素进行制取。首先选用酸碱法,从黑色长牡蛎中提取出天然长牡蛎黑色素,将其溶于氢氧化钠溶液中,利用超声细胞破碎仪进行处理,用盐酸中和至中性,离心取上清并干燥后得到一种黑色可溶性固体;再将该黑色固体分别用激光粒度测试仪、红外光谱仪和紫外光谱扫描仪进行检验后,发现天然长牡蛎黑色素经过超声降解处理后,颗粒粒度大幅下降,其溶解性大幅提高。红外光谱则显示出1630nm左右有明显的峰值,说明超声降解处理并未破坏真黑色素吲哚环等官能团;紫外吸光图谱的比较则显示天然长牡蛎黑色素破碎后,吸收峰仍然出现在210nm左右,但吸收值明显升高,说明超声降解法可以将难溶于水的天然长牡蛎黑色素降解为可溶于水的小颗粒可溶性黑色素,从而提高了紫外吸收值。可溶性牡蛎黑色素的制备成功对推动牡蛎黑色素的功能研究具有重要意义。     

Response of phytoplankton to multispecies mariculture:a case study on the carrying capacity of shellfish in the Sanggou Bay in China

A muhispecies model for shellfish polycuhure in the Sanggou Bay in China used for large-scale long-line cultivation of the Chinese scallop Chlamysfarreri, the Pacific oyster Crassostrea gigas and the kelp Laminaria japonica is presented. The model includes key physical processes which are the transports of matter at the system boundary, and the main biological process that is the primary production and nutrients release from the bottom. By the model, the seasonal fluctuations of phytoplankton biomass and dissolved inorganic nitrogen（DIN） in 1994 are simulated. Furthermore, if the kelp culture scale is kept constant and the Chinese scallop and the Pacific oyster culture scales are adjusted, virtual shellfish farms are funded and responses of phytoplankton to the largescale shellfish culture are simulated. According to these simulated results, the room limitation, and the hypothesis that shellfish will not grow well if the phytoplankton biomass is less than 8.2 mg/m^3 , the expandable multiple of scallop culture k and that of oyster culture y are determined as k = -0.276 5y ＋4.690 5 and 0.133 3k ＋0.006 6y≤0.667 5, where, k （ or y） is equal to 1, the culture scale of scallop （ or oyster） is 8.8 x 109 individuals （or 66 ha, with a density of 59 ind./m^2 ）, and the kelp culture scale is 3 300 ha with a density of 12 ind./m^2.     

Short-Range Prediction Experiments with Operational Data Assimilation System for the Kuroshio South of Japan

The short-range (one month) variability of the Kuroshio path was predicted in 84 experiments (90-day predictions) using a model in an operational data assimilation system based on data from 1993 to 1999. The predictions started from an initial condition or members of a set of initial conditions, obtained in a reanalysis experiment. The predictions represent the transition from straight to meander of the Kuroshio path, and the results have been analyzed according to previously proposed mechanisms of the transition with eddy propagation and interaction acting as a trigger of the meander and self-sustained oscillation. The reanalysis shows that the meander evolves due to eddy activity. Simulation (no assimilation) shows no meander state, even with the same atmospheric forcing as the prediction. It is suggested therefore that the initial condition contains information on the meander and the system can represent the evolution. Mean (standard deviation) values of the axis error for all 84 cases are 13, 17, and 20 (10, 10, and 12) km, in 138.5°E, in the 30-, 60-, and 90-day predictions respectively. The observed mean deviation from seasonal variation is 30 km. The predictive limit of the system is thus about 80 days. The time scale of the limit depends on which stage in the transition is adopted as the initial condition. The gradual decrease of the amplitude in a stage from meander to straight paths is also predicted. The predictive limit is about 20 days, which is shorter than the prediction of the opposite transition. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effects of silver nanoparticles on oyster embryos

Nanoparticles may be introduced into aquatic environments during production processes and also as a result of release following their use in various commercial formulations and biologic applications. Filter-feeding bivalve mollusks such as oysters are valuable model species for characterizing nanoparticle bioavailability and interactions with basic cellular processes. The adults release their gametes into the environment, so their embryos and larvae are also likely targets of nanoparticles. The purpose of these studies was to characterize the toxicity of metal nanoparticles on embryonic development of oysters, Crassostrea virginica and to compare the relative sensitivity of embryos to adults. Newly-fertilized oyster embryos were exposed to silver nanoparticles (AgNP) and then the percent normal development after 48 h was assessed. Studies were conducted with adult oysters in which they were also exposed to AgNP for 48 h, and the effects on lysosomal destabilization were determined. The expression of metallothionein (MT) gene expression was also assessed in both embryos and adults. Adverse effects on embryonic development were observed at concentrations similar to those that caused both statistically and biologically significant effects on lysosomal destabilization of adults. Significant increases in MT mRNA levels were observed in both embryos and adult oysters, and MT levels were highly induced in embryos. While we do not know whether the toxicity and gene expression responses observed in this study were due to the nanoparticles themselves or the Ag ions that dissociated from the nanoparticles, these kinds of basic studies are essential for addressing the potential impacts of nanoengineered particles on fundamental cellular processes as well as aquatic organisms.