Rickettsia-like organism infection associated with mass mortalities of blood clam, Tegillarca granosa, in the Yueqing Bay in China

A series of mass mortalities of the cultured blood clam, Tegillarca granosa, occurred in the Yueqing Bay of China from 2005 to 2009. An obligate intracellular prokaryote, designated as rickettsia-like organism (RLO), was frequently found in the moribund or dead blood clam sample during ultrastructural examination. These organisms were usually round, ellipsoid or occasionally dumbbell-shaped, ranged from approximately 0.28 to 0.71 μm in size and had a trilaminar cell wall. Two reproductive modes of organisms, transverse binary fission and budding, were observed. The organisms were able to form eosinophilic inclusions. Most inclusions were found within epithelial and connective tissues of the mantle, gills and digestive tube. The biological and morphological characteristics indicate that these organisms may belong to the family Rickettsiaceae. RLOs exhibited significant pathogenicity. Cytopathological examinations revealed extensive necrosis and destruction in the infected cell. The degree of tissue destruction was positively related to the number of RLO inclusions in the tissues, and the cytopathological effects were positively related to the number of intracellular RLO. RLOs and their inclusions were discovered throughout different disease areas and in different time periods. The infection intensity of the RLOs was positively correlated with the mortality rate of clams. Therefore, RLO infection might be associated with mass mortalities of cultured blood clams in the Yueqing Bay.     

Effects of tributyltin at environmental levels on monooxygenase system of digestive gland in hard clam Meretrix meretrix

The effects on ethoxyresorufin O-deethylase （EROD）, NADPH-cytochrome c reductase and NADH cytochrorne b5 reductase activities of digestive gland in Meretrix meretrix exposed to tributyltin （TBT） at environmental levels （0.1,1.0,10.0 ng/dm as stannum concentration）,in experimental condition, were evaluated. The EROD, NADH cytochrorne b5 reductase activities were significantly inhibited after exposure to 10.0 ng/dm^3 TBT for 8 and 20 d, the NADPH cytochrorne c reductase activities were significantly inhibited after exposure to 0.1,1.0 and 10.0 ng/dm TBT for 8 d and to 1.0 and 10.0 ng/dm for 20 d, as compared with the matched control, while NADH cytochrorne b5 reductases and NADPH cytochrome c reductase activities were induced after exposure to 10.0 ng/dm^3 TBT for 2 d. The EROD activity in the 10 ng/dm^3 group,and the NADH cytochrome b5 reductases activities in 1.0 and 10.0 ng/dm groups, were significantly induced when transferred to clean recovery tanks for 7 d. The three enzymic activities in the clams exposed to TBT were recovered to the level corresponding to that of the control group after transfer to clean recovery tanks for 20 d. NADPH cytochrome c reductase activity in Meretrix meretrix seems to be more sensitive to exposure of TBT than that of the EROD and NADH cytochrome b5 reductases. The results suggest that induction and inhibition by TBT to the monooxygenase system enzymic activity in Meretrix meretrix would simultaneously exist. The enzymic activities were inhibited by exposure for a long time. The results suggest that inhibition of the monooxygenase system should be an indication of the exposure to environmentally relevant concentrations of TBT for a long time.     

Enhanced a novel β-agarase production in recombinant Escherichia coli BL21(DE3) through induction mode optimization and glycerol feeding strategy

Agarases are hydrolytic enzymes that act on the hydrolysis of agar and have a broad range of applications in food,cosmetics and pharmaceutical industries. In this study, a glycerol feeding strategy based on induction mode optimization for high cell density and β-agarase production was established, which could effectively control acetate yield. First, exponential feeding strategy of glycerol with different overall specific growth rates(μ) was applied in the pre-induction phase. The results showed that the low μ(μ=0.2) was suggested to be the optimal for cell growth and β-agarase production. Second, the effects of induction temperature and the inducer concentration on cell growth and β-agarase production were investigated in the post-induction phase. When induced by isopropyl-β-d-thiogalactoside(IPTG), the strategy of 0.8 mmol/L IPTG induction at 20℃ was found to be optimal for β-agarase production. When cultivation was induced by continuous lactose feeding strategy of 1.0 g/(L·h), the β-agarase activity reached 112.5 U/mL, which represented the highest β-agarase production to date.Furthermore, the β-agarase was capable of degrading G. lemaneiformis powder directly to produce neoagarooligosaccharide, and the hydrolysates were neoagarotetraose(NA4) and neoagarohexaose(NA6). The overall research may be useful for the industrial production and application of β-agarase.     

Enzymatic properties of UFE, a novel marine fibrinolytic enzyme

A novel potent protease, Urechis unicinctus fibrinolytic enzyme (UFE), was firstly discovered. The enzymatic properties of UFE were further investigated. As a low molecular mass protein, UFE appeared to be very stable to heat and pH. When temperature was below 50 ℃, the remnant enzyme activity remained almost unchanged, but when temperature was raised to 60 ℃, the remnant enzyme activity began to decrease rapidly. UFE was quite stable in the range of pH value from 3 to 12, especially in slightly alkaline pH value. Mn2 , Cu2 and Fe2 ions were activators of UFE, while Fe3 and Ag ions were inhibitors of UFE. Fe2 ion along with Fe3 ion might regulate UFE activity in vivo. The optimum pH and temperature of UFE were about 8 and 50 ℃, respectively. Other characteristics of this enzyme were also studied. Systematic research results are significant when UFE is applied for medical and industrial purposes.     

Dynamic Behavior and Deformation Analysis of the Fish Cage System Using Mass-Spring Model

Fish cage systems are influenced by various oceanic conditions, and the movements and deformation of the system by the external forces can affect the safety of the system itself, as well as the species of fish being cultivated. Structural durability of the system against environmental factors has been major concern for the marine aquaculture system. In this research, a mathematical model and a simulation method were presented for analyzing the performance of the large-scale fish cage system influenced by current and waves. The cage system consisted of netting, mooring ropes, floats, sinkers and floating collar. All the elements were modeled by use of the mass-spring model. The structures were divided into finite elements and mass points were placed at the mid-point of each element, and mass points were connected by springs without mass. Each mass point was applied to external and internal forces, and total force was calculated in every integration step. The computation method was applied to the dynamic simulation of the actual fish cage systems rigged with synthetic fiber and copper wire simultaneously influenced by current and waves. Here, we also tried to find a relevant ratio between buoyancy and sinking force of the fish cages. The simulation results provide improved understanding of the behavior of the structure and valuable information concerning optimum ratio of the buoyancy to sinking force according to current speeds.     

Key genes expression of reductive tricarboxylic acid cycle from deep-sea hydrothermal chemolithoautotrophic Caminibacter profundus in response to salinity, pH and O2

CO2 fixation pathway of Caminibacter profundus, a chemolithoautotrophic -Proteobacteria from deep-sea hydrothermal vent, was determined and characterized by genetic and enzymatic analyses. Gene expression of key enzymes for CO2 fixation in response to salinity, pH and O2 in Medium 829 were also investigated. The results demonstrate that C. profundus contained aclB, porA and oorA, the genes encoding key enzymes of reductive tricarboxylic acid (rTCA) cycle. However, genes fragments of cbbL and cbbM encoding key enzyme of Calvin cycle were not recovered. Key enzymatic activities of ATP citrate lyase (ACL), pyruvate: ferredoxin oxidoreductase (POR) and 2-oxoglutarate: ferredoxin oxidoreductase (OOR) were also present in C. profundus. The combination of genetic and enzymatic analyses confirm that C. profundus adopted rTCA cycle for carbon assimilation. The results of aclB and oorA relative expressions of C. profundus demonstrate that the ranges of environmental factors for high genes expression were sea salt 3.0%-5.0% (optimum 3.0%), pH 5.0-6.5(optimum pH 6.5), anaerobic to microaerobic conditions (optimum 1.0% O2 ). Gene expression patterns under different conditions show similar patterns with bacterial growth, revealing that key rTCA cycle genes provided molecular basis for bacterial growth and propagation. Our results suggest that C. profundus could regulate key genes of rTCA cycle for carbon assimilation and energy metabolism in response to environmental fluctuations in hydrothermal vent.     

Components of Decellwall Enzymes of Three Sea Snails-Seaweed''s Structural Polysaccharidases and Trehalase

Enzymic mixtures were extracted from digestive organs of three sea snails. All of the three mixtures can decompose the cell walls of the seaweeds. The present note reports another five enzymic components in the three mixtures. These enzymes can hydrolyze algin, carrageenan, agar, fucoidan and trehalose. The levels of these enzymic activities are all in the order: Lunella cornata coreensis>Monodonta labio>Purpura     

Phylogenetic analysis and biological characteristic tests of marine bacteria isolated from Southern Ocean (Indian sector) water

Fifty-seven bacteria were isolated from Southern Ocean(Indian sector) water samples which were collected from different latitude and longitude of the ocean. All the isolates were able to grow at 4°C, 20°C, 37°C and tolerable Na Cl concentration up to 13.5%(w/v). 29 out of 57 isolates were identified using 16 S rDNA amplification and the sequences were submitted to National Center for Biotechnology Information(NCBI). All the isolates were classified by using Ribosomal Database Project(RDP) and found that isolates belongs to Proteobacteria and Bacteriodes. The average G+C content was 56.4%. The isolates were screened for the presence of extracellular enzymes, viz. amylase, catalase, urease, esterase, lipase and protease. The disc diffusion method is used to screen antibiotic production by the isolates against four pathogenic bacteria, viz. Salmonella typhimurium(NCIM 2501),Staphylococcus aureus(NCIM 2122), Bacillus subtilis(NCIM 2193), and Pseudomonas aeruginosa(NCIM 2036).Nine out of 29 were found to be antibiotic producer.     

Effects of tributyltin at environmental levels on monooxygenase system of digestive gland in hard clam Meretrix meretrix

The effects on ethoxyresorufin O-deethylase (EROD), NADPH-cytochrome c reductase and NADH cytochrome b5 reductase activities of digestive gland in Meretrix meretrix exposed to tributyltin (TBT) at environmental levels (0.1,1.0, 10.0 ng/dm3 as stannum concentration), in experimental condition, were evaluated. The EROD, NADH cytochrome b5 reductase activities were significantly inhibited after exposure to 10.0 ng/dm3 TBT for 8 and 20 d, the NADPH cytochrome c reductase activities were significantly inhibited after exposure to 0.1,1.0 and 10.0 ng/dm3 TBT for 8 d and to 1.0 and 10.0 ng/dm3 for 20 d, as compared with the matched control, while NADH cytochrome b5 reductases and NADPH cytochrome c reductase activities were induced after exposure to 10.0 ng/dm3 TBT for 2 d. The EROD activity in the 10 ng/dm3 group, and the NADH cytochrome b5 reductases activities in 1.0 and 10.0 ng/dm3 groups, were significantly induced when transferred to clean recovery tanks for 7 d. The three enzymic activities in the clams exposed to TBT were recovered to the level corresponding to that of the control group after transfer to clean recovery tanks for 20 d. NADPH cytochrome c reductase activity in Meretrix meretrix seems to be more sensitive to exposure of TBT than that of the EROD and NADH cytochrome b5 reductases. The results suggest that induction and inhibition by TBT to the monooxygenase system enzymic activity in Meretrix meretrix would simultaneously exist. The enzymic activities were inhibited by exposure for a long time. The results suggest that inhibition of the monooxygenase system should be an indication of the exposure to environmentally relevant concentrations of TBT for a long time.     

Rates of bacterial sulfate reduction and their response to experimental temperature changes in coastal sediments of Qi’ao Island, Zhujiang River Estuary in China

Subtropical sediment cores(QA09-1 and QA12-9) from the coastal zone of Qi'ao Island in the Zhujiang River Estuary were used to determine the rates of sulfate reduction and their response to experimental temperature changes. The depth distribution of the sulfate reduction rates was measured from whole-core incubations with radioactive tracer 35SO42-, and peaks of 181.19 nmol/(cm3·d) and 107.49 nmol/(cm3·d) were exhibited at stations QA09-1 and QA12-9, respectively. The profiles of the pore water methane and sulfate concentrations demonstrated that anaerobic oxidation of methane occurred in the study area, which resulted in an increase in the sulfate reduction rate at the base of the sulfate-reducing zone. Meanwhile, the sulfate concentration was not a major limiting factor for controlling the rates of sulfate reduction. In addition, the incubation of the sediment slurries in a block with a temperature gradient showed that the optimum temperature for the sulfate reduction reaction was 36℃. The Arrhenius plot was linear from the lowest temperature to the optimum temperature, and the activation energy was at the lower end of the range of previously reported values. The results suggested that the ambient temperature regime of marine environments probably selected for the microbial population with the best-suited physiology for the respective environment.     

海带雌性原生质体的分离与培养

于1993年1月 - 1993年4月，为了制备遗传性纯一的基因工程受体，用酶法从海带雌配子体单性生殖系分离雌性单倍体原生质体。从皱纹盘鲍消化腺及消化器官提取出鲍酶，在25°C下、1％（W／V）的鲍酶与2％（W／V）的纤维素酶（Onozuka R－10）合用能有效解离单细胞和原生质体。海带雌性原生质体经2d培养再生了细胞壁。     

海藻工具酶研究Ⅱ.褐藻酸酶的制备、性质及对裙带菜细胞的解离研究

褐藻酸降解菌埃氏交替单胞菌(Alteromonas espejiana)菌株Al01,通过发酵培养制备褐藻酸酶.该酶作用的最适pH为7.0,最适温度为40℃,最适底物浓度为1%～2%;在离子浓度为0.5mmol/dm3时,Mn2+对酶促反应稍有促进作用,Ca2+、Hg2+则有明显的抑制作用.该酶用于裙带菜单细胞和原生质体解离时,以酶液组成为褐藻酸酶1%、纤维素酶1%,45×10-3的NaC1为渗透剂,酶解温度25℃,pH7.0,酶解3-4h效果最好.     

温度和pH 对杂色鲍消化酶活力的影响

采用酶学分析方法研究了温度和pH对杂色鲍(Haliotis diversicolor Reeve)肝胰腺和消化道中胃蛋白酶、类胰蛋白酶、淀粉酶和纤维素酶4种主要消化酶活力的影响。结果表明,在设定的温度和pH范围内,杂色鲍各消化酶的活力均随着温度或pH的升高呈现先升后降的变化趋势。其中,肝胰腺和消化道中胃蛋白酶的最适温度均为50℃,适宜pH分别为2.6～3.0、2.6～3.4;类胰蛋白酶在肝胰腺中的最适温度为40℃、适宜pH为8.6～9.0,在消化道中的最适温度为50℃,适宜pH为8.2;肝胰腺和消化道中淀粉酶的最适温度分别为40、30℃,最适pH分别为6.8、7.2;纤维素酶在肝胰腺中的适宜温度为40～50℃,最适pH为5.2,在消化道中的最适温度为40℃,适宜pH为4.8。在最适温度和pH范围内,肝胰腺中纤维素酶、淀粉酶活力均显著高于消化道中,具有器官特异性,而胃蛋白酶和类胰蛋白酶活力在肝胰腺和消化道中差异不显著,无器官特异性。同种消化器官内,各消化酶的活力也存在差异。     

九孔鲍褐藻酸酶、琼脂酶及纤维素酶的提取纯化

采用（NH4）2SO4分段盐析和葡聚糖凝胶SephadexG-100柱层析纯化技术，从九孔鲍Haliotis diversicolor supertexta内脏器官中提取纯化褐藻酸酶、琼脂酶及纤维素酶，结果表明，在（NH4）2SO4分段盐析纯化中，褐藻酸酶和纤维素酶的最适分离饱和度为60%，而琼脂酶为70%，分段盐析的提纯倍数为（以粗酶提取液为参照）褐藻酸酶13.3 ,琼脂酶8.7和纤维素酶10.9。葡聚糖凝胶SephadexG-100层析分离过程中，褐藻酸酶，琼脂酶和纤维素酶的比活力高峰分别出现在洗脱液的64，48和80ml处，提纯倍数分别为褐藻酸酶80.9，琼脂酶68.0及纤维素酶15.2，上述提纯方法的研究结果将为这3种酶性质的进一步研究以及作为工具酶制剂产品的开发提供了工艺技术基础。     

粤东养殖区分离的2株海洋弧菌及其胞外产物对皱纹盘鲍致死毒性的初步分析

2011年秋,福建和广东地区养殖的皱纹盘鲍(Haliotis discus hannai)相继暴发死亡。同年12月,我们从广东汕头病区取样分离得到2株弧菌,其中bb3为溶藻弧菌(Vibrio alginolyticus), bb4为哈氏弧菌(V. harveyi)。本研究以bb3、bb4作为供试菌株对1龄皱纹盘鲍进行侵染实验,获得如下结果：20℃条件下,供试菌及其胞外产物粗提液注射处理均可致受试鲍死亡,浓度2.5×107 cfu/mL 的 bb3与2.0×107cfu/mL的bb4悬液注射可分别引发43.33%和78.02%的受试鲍死亡;供试菌株的致死毒性与注射弧菌的总量正相关,与受试幼鲍的壳长、全湿重负相关;注射或浸泡-创伤两种处理方式均可导致受试鲍死亡,并且因注射供试弧菌而死亡的幼鲍软体部组织被健康鲍取食后也可引发取食者死亡;从侵染实验死亡的受试鲍软体部组织中可重新分离得到这2种供试弧菌。上述结果表明,溶藻弧菌 bb3和哈氏弧菌bb4是皱纹盘鲍的病原菌。药敏试验表明, bb3、bb4均已对抗生素具有一定程度的耐药性,在测试的20种抗生素中,仅有头孢曲松和丁胺卡那霉素是这2株弧菌同时高度敏感的抗生素,但bb3和bb4却对8种抗生素同时表现出抗性。     

金属离子和pH值对九孔鲍几种消化酶活力的影响

研究了金属离子对九孔鲍(Haliotis diversicolor supertepla)褐藻酸酶、琼脂酶及纤维素酶活力的影响。结果表明，3种酶的最适pH不同。分别为褐藻酸酶8．8、琼脂酶4．2、纤维素酶4．5-5．0。MgSO4为褐藻酸酶的激活剂、BaCl2为琼脂酶的激活剂、MnCl2是纤维素酶的激活剂。     

北冰洋海单胞菌β-D-半乳糖苷酶的异源表达及酶学特性研究

初筛表明,一株分离自北极加拿大海盆海冰心芯样品的海单胞菌(Marinomonas sp.BSi20584)具有较高的β-D-半乳糖苷酶活性,为了研究清楚其酶学性质,将经hiTAIL-PCR扩增得到的β-D-半乳糖苷酶基因(galt)与pET-28a(+)原核表达载体结合,转入大肠杆菌BL21(DE3)。经IPTG诱导后对重组β-D-半乳糖苷酶(GALT)的表达条件进行了优化,采用金属螯合亲和层析技术制备纯酶,并对重组GALT的酶学性质进行了研究。结果显示,重组酶的最适诱导温度为20℃,在IPTG浓度为0.07mmol/L时诱导22h后,酶活和产酶量达到最大值。GALT单体分子量约为6.6×104 g/mol,天然酶为同源三聚体。GALT最适作用温度为35℃,其热稳定性较好,在60℃处理5h后,仍可保持50%以上的相对活性。GALT的最适作用pH为9.0,在pH为6.0~11.0范围内比较稳定。GALT的最适NaCl浓度为0.5mol/L,对盐度具有较高的耐受性。Mg2+、K+、DTT和EDTA对酶活不具有显著影响,而Mn2+、Fe2+对酶活有促进作用,Zn2+和L-谷胱甘肽对酶活有抑制作用。GALT对Galβ1-4GlcNAc具有水解作用,而对Galβ1-3GalNAc和Galβ1-3GlcNAc糖苷键型没有水解能力。本研究实现了海单胞菌属菌株的β-D-半乳糖苷酶基因在大肠杆菌系统中的高效表达,并系统研究了重组酶的酶学特性,为后续开展该酶的代谢适应性和潜在应用研究提供详细的酶学数据基础。     

单体牡蛎诱导变态的研究

为了得到壳型规则、大小均一的单体牡蛎,作者以葡萄牙牡蛎(Crassostrea angulata)和长牡蛎(Crassostrea gigas)眼点幼虫为材料,从肾上腺素(EPI)浓度梯度、处理时间梯度和眼点幼虫密度梯度3个因素诱导产生不固着变态的单体牡蛎。结果表明,葡萄牙牡蛎用EPI处理24 h的最适浓度为5×10–4 mol/L,不固着变态率为72.8%,在该最适浓度下,最佳处理时间为12 h,不固着变态率为82.7%。长牡蛎用EPI处理6 h的最适浓度为5×10–5 mol/L,不固着变态率为53.2%,在该最适浓度下,最佳处理时间为8h,不固着变态率为56.8%,眼点幼虫密度在80个/m L以下EPI处理效果没有显著性差异。对长牡蛎幼虫进行后续生长测定,结果显示EPI处理组幼虫的壳长、壳高和存活率要高于对照组,表明EPI可能促进牡蛎幼虫变态长出次生壳并提高其生存能力。     

微拟球藻原生质体制备与再生

综合考虑酶混合液处理时间和初始细胞密度两个因素,选取终浓度为4%半纤维素酶和2%崩溃酶的酶混合液对一株海洋经济微藻——微拟球藻(Nannochloropsis oculata)进行原生质体的制备与再生,Calcofluor White染料染色可在荧光显微镜下观察到完整的原生质体细胞。实验结果表明:同一初始密度藻细胞酶处理1～3 h制备率较高;酶处理相同时间较高初始密度(2×107～3×107cells/mL)的藻细胞制备率较高,并且原生质体在再生培养基上可再生,生长趋势与野生型细胞一致。考虑到酶处理时间过长或者密度过大会对原生质体的再生产生影响,本实验选择最适酶处理时间为1h,初始细胞密度为2×107cells/mL。     

普里兹湾附近绕极深层水和底层水及其运动特征

利用中国第15次南极科学考察科学考察队的CTD全深度观测资料(1998年11月至1999年2月),分析并讨论了普里兹湾以北的南大洋海域内,绕极深层水(CDW)和南极底层水(AABW)的物理特性及其空间分布.同时还与历史上其他学者的发现进行了比较.指出了在研究海域内,CDW在100～2000m之间从北向南扩展,其高温核(t>1.2℃)和高盐核(S>34.7)在75°E断面上最为深厚,向南扩展得最远;而AABW则在2500m以深由陆坡底部向北扩展,σ_θ>27.875的高密度水体在70°E断面上最为深厚,向北扩展得最远.此外还通过实测的CTD资料证实了CDW和AABW的经向环流特征,以及它们与迪肯流环(Deaconcell)、亚极地流环和深层流环的一致性.