腐败希瓦氏菌噬菌体的分离纯化和生物学性质

以腐败希瓦氏菌Sp225为宿主菌,采用噬菌斑法和双层平板法分离纯化腐败希瓦氏菌噬菌体Spp001,并对其生物学性质进行研究。经分离纯化得到的Spp001裂解性好,噬菌斑清晰、大小一致。经鉴定,Spp001为长尾噬菌体科,核酸为DNA。对其性质研究表明,Spp001具有较好的热稳定性,且耐碱不耐酸,其最佳感染复数为10,一步生长曲线显示该噬菌体感染宿主菌潜伏期约为10min,爆发期约为20min,裂解量为98。研究首次分离纯化了腐败希瓦氏菌的噬菌体Spp001并研究其生理特性,对进一步研究抑制冷藏水产品的腐败具有重要意义。     

一株希瓦氏菌低温噬菌体的分离及特征研究

采用双层平板法从新疆东帕米尔高原喀拉库勒湖中分离纯化低温噬菌体,并对其特征进行研究。结果分离得到1株烈性低温噬菌体,命名为KLP17,其宿主菌KLB17经16SrRNA基因序列分析鉴定为Shewanella菌株。KLP17裂解性好,可在双层平板上感染宿主菌KLB17形成直径2-4mm的圆形、透明噬菌斑。电镜下其颗粒形态呈头尾型,头部为正多面体对称,直径约88nm,收缩性尾长约160nm,属肌尾噬菌体科(Myoviridae)。噬菌体KLP17对高温敏感,耐碱不耐酸,对氯仿和异丙醇均不敏感,但对SDS和蛋白酶K十分敏感,推测其衣壳组分为蛋白质而无脂质包被。KLP17最佳感染复数为0.01,感染宿主菌KLB17的潜伏期、裂解期分别为55min和30min,裂解量为27。酶切结果显示其基因组为dsDNA,大小约67~69kb,研究结果不仅为进一步探究微生物适冷机制积累重要素材,而且为深入解析低温噬菌体与宿主菌及环境的互作关系提供数据参考。     

副溶血弧菌胶体金快速检测试纸的研制及应用

副溶血弧菌(Vibrio parahaemolyticus)是海水养殖动物弧菌病的主要病原菌之一。为实现对该菌的现场快速检测,本文以制备的兔抗副溶血弧菌多克隆抗体作为金标抗体,提取副溶血弧菌的外膜蛋白、鞭毛蛋白、胞外产物和全菌蛋白,以不同浓度的4种抗原蛋白作为4条检测线(T1~T4),特别在T4线设置全菌蛋白,基于竞争免疫层析原理研制出副溶血弧菌胶体金快速检测试纸。使用该试纸检测了副溶血弧菌(V.parahaemolyticus)、鳗弧菌(V.anguillarum)、溶藻弧菌(V.alginolyticus)、哈维氏弧菌(V.harveyi)和鱼肠道弧菌(V.ichthyoenteri),检测结果表明其能够准确鉴别副溶血弧菌感染,排除其他菌的交叉反应干扰。副溶血弧菌快速检测试纸的最低检测限为5×105 cfu·mL^-1,感染副溶血弧菌的牙鲆组织的检测结果与ELISA结果一致。本文研制的试纸具有较高的特异性与准确性且操作简单,为现场快速检测副溶血弧菌提供了有力工具。     

两株溶藻弧菌噬菌体的生理特性和基因组研究

利用噬菌体治疗水产养殖细菌病害具有专一性强、自我增殖能力强等优点, 可以有效减少对环境的污染。为了获得可防治水产病原体溶藻弧菌(Vibrio alginolyticus)的生物杀菌剂, 本研究以Vibrio alginolyticus ATCC 17749^T为宿主, 从福建省东山岛的对虾养殖场与水产市场污水环境中分离得到两株巨型噬菌体vB_ValM_R10Z和vB_ValM_R11Z, 并分别从噬菌体形态、宿主范围、生命周期和基因组信息等方面进行了鉴定和分析。实验结果显示, 噬菌体R10Z和R11Z具有相似的噬菌斑形态和电镜形态, 属于肌尾噬菌体; 除宿主之外, 噬菌体R10Z和R11Z均能侵染另一株欧文氏弧菌Vibrio owensii JL3186; 两株噬菌体均对氯仿不敏感, 表明其衣壳蛋白不含脂类物质; 噬菌体R10Z和R11Z的潜伏期均为20min, 但裂解量却相差一倍, 分别为45PFU·cell^-1和114PFU·cell^-1; 两株噬菌体的基因组大小分别为247167bp和246831bp, G+C含量分别为41.30%和41.33%, 两者基因组相似性达99.45%, 均未检测出毒力基因与抗药性基因; 系统发育分析显示两株噬菌体均属于Myoviridae(科), Tevenvirinae(亚科), Schizotequatrovirus(属), 同源基因在环境中分布广泛。两株噬菌体性状优良, 在水产病害的噬菌体治疗领域具有重要的研究价值和潜在的应用价值。     

文蛤副溶血弧菌间接 ELISA检测技术的研究

从患病文蛤组织内分离出一株细菌,回归感染试验证明是文蛤的致病菌,经鉴定为副溶血弧菌,灭活后(56 ℃,1 h)制成菌苗,用该菌苗制备兔抗血清,以辣根过氧化酶标记的羊抗兔血清(HRP-IgG)为酶标二抗,对副溶血弧菌进行间接ELISA快速检测.结果表明:应用间接 ELISA 技术检测副溶血弧菌具有较高的灵敏度,其最低检测极限为 1 × 105 cfu / mL.患病文蛤内脏团中副溶血弧菌的检出率为 80 %,无病症带菌文蛤中的检出率为 15 %,海水中副溶血弧菌的浓度低于最低检测极限.表明间接 ELISA检测法不仅可以用于发病文蛤的快速检测,而且能够检测出无病症的带菌的文蛤,为有效预防副溶血弧菌病的暴发提供了检测手段.     

噬菌体防治皱纹盘鲍脓疱病的研究

于１９９３年６月在大连太平洋海珍品养殖公司的１２个采集点分离到河流弧菌的Ⅱ的噬菌体,依据过量噬菌体可以全部裂解其宿主菌河流弧菌Ⅱ的特性,用噬菌体对皱纹盘鲍脓疱病进行了生物防治研究。结果表明,使用一定浓度的噬菌体可以有效地治疗或推迟脓疱病引起的鲍死亡,可将鲍的成活率提高５０％以上。探讨了在噬菌生物防治中遇到的难题及解决办法、噬菌体分离规律等问题。     

分子鉴定方法研究大亚湾水体弧菌种类变化

借助分子鉴定方法监测大亚湾水体中弧菌Vibrio种类季节性动态的变化规律.通过增菌培养、菌株分离,在224份海水中共分离出弧菌368株,并用分子生物学辅助生化鉴定方法鉴定弧菌菌株.结果表明,在大亚湾海域水体中鉴定的弧菌种类有溶藻弧菌Vibrio alginolyticus、副溶血弧菌V.parahaemolyticus、哈氏弧菌V.harveyi和创伤弧菌V.vuinificus,没有检测到霍乱弧菌V.cholerae、拟态弧菌V.mimicus、河流弧菌V.fluvialis和霍利斯弧菌V.hollisae.溶藻弧菌和副溶血弧菌为优势菌群,分别占弧菌总数的27.99%和21.74%.     

养殖大黄鱼(Pseudosciaena crocea)3种致病弧菌的分子鉴定及其系统发育学分析

从患病养殖大黄鱼分离到3株病原菌H040823-1、H050704-1、H050815-1,经常规生理生化鉴定均属于弧菌属的种类,API20E快速鉴定菌株H040823-1为副溶血弧菌(Vibrio parahaemolyti-cus),菌株H050815-1为溶藻弧菌(V.alginolyticus),菌株H050704-1不在API20E鉴定谱内。为了进一步确定其分类地位,测定了3株病原菌的16S rRNA和HSP60(heat shock protein,HSP60)基因部分序列。16S RNA基因系统进化分析表明,3株病原菌与副溶血弧菌、哈维氏弧菌、溶藻弧菌亲缘关系较近,相互之间同源性均大于96.9%,差异不明显。HSP60基因序列分析表明,菌株H040823-1、H050704-1、H050815-1的HSP60基因序列分别与V.parahaemolyticus(AF230951)、V.harveyi(EU036994)、V.alginolyticus(DQ664545)的同源性最高,分别为96.7%、99.8%和98.0%,而与其他弧菌HSP60基因的同源性均低于91.9%,3株病原菌相互之间同源性低于92.3%,差异显著。HSP60基因构建的系统进化树表明,H040823-1、H050704-1、H050815-1分别与V.parahaemolyticus、V.harveyi、V.alginolyticus聚类。综合以上结果,菌株H040823-1、H050704-1、H050815-1可分别鉴定为副溶血弧菌、哈维氏弧菌、溶藻弧菌。结果表明,HSP60基因比16S rRNA基因更适合用于海水鱼类致病性弧菌种间的分类研究。     

一株副溶血弧菌D3112致病性和抗药性的表征

副溶血弧菌广泛分布于海洋环境中,虽然大多数环境菌株并非致病菌,但它们常常携有毒力基因,从而具有潜在的致病性。本文从黄海(山东青岛)海水中分离到一株副溶血弧菌D3112,对其进行了全基因组测序、溶血实验和人工感染实验等。基因组测序分析发现D3112并不含有副溶血弧菌的致病性标志溶血素基因tdh和trh,这与PCR检测结果一致,但含有其他溶血素基因以及多种毒力相关基因。生物学实验显示,该菌可产生明显的溶血现象,而且具有蛋白酶、明胶酶、脂酶和淀粉酶活性,但是缺少卵磷脂酶活性。人工感染实验表明副溶血弧菌D3112具有致病性,感染斑马鱼的半致死剂量约为5×105CFU。药物敏感实验证明了D3112具有多重耐药性。本文对海洋环境中的副溶血弧菌D3112同时开展了基因型和表型研究,为准确评价环境细菌的潜在致病性提供了有用的数据信息。     

一株副溶血弧菌qdfsVp001的分离与鉴定

用弧菌选择从牙鲆饲育水中分离出1株细菌qdfsVp001,对该菌在培养基上的菌落形态、生理生化特性及其16SrDNA基因序列进行研究,结果表明该菌在TCBS平板上呈现大的蓝绿色菌落,在弧菌显色培养基上呈现紫色菌落,其生理生化特性具有副溶血弧菌的典型特征。16SrD-NA基因序列及系统发育树分析表明,该菌与弧菌的亲缘关系最近,其中尤与副溶血弧菌最为相似。结合qdfsVp001的形态、生理生化及16SrDNA基因序列分析结果,确定该菌株是1株副溶血弧菌。     

Cloning and expression analysis of the chloroplast fructose- 1,6-bisphosphatase gene from Pyropia haitanensis

Fructose-1,6-bisphosphatase(FBPase) is one of the key enzymes in Calvin circle and starch biosynthesis. In this study, the full-length of cpFBPase gene from Pyropia haitanensis was cloned by using rapid amplification of cDNA ends(RACE) technology. The nucleotide sequence of PhcpFBPase consists of 1 400 bp, including a 5′ untranslated region(UTR) of 92 bp, a 3′?UTR of 69 bp, and an open reading frame(ORF) of 1 236 bp, which can be translated into a 412-amino-acid putative peptides with a molecular weight of 44.3 kDa and a theoretical pI of 5.23. Multiple sequence alignment indicated that the protein belonged to the chloroplast FBPase enzyme. Phylogenetic analysis showed that the protein assembled with the cpFBPase of a thermal tolerant unicellular red micro-algae Galdieria sulphuraria. Expression patterns analyzed by qRT-PCR revealed that the expression of PhcpFBPase gene in the thallus phage was 7-fold higher than in the conchocelis phage, which suggested the different mechanisms of inorganic carbon utilization among the different life phages of P. haitanensis. And the different response modes of PhcpFBPase mRNA levels to high temperature and desiccation stress indicated that PhcpFBPase played an important role in responsing to abiotic stress.     

Seasonal Variations in Abundance and Size Composition of the Lobate Ctenophore Bolinopsis Mikado (Moser) in Tokyo Bay, Central Japan

Surveys of the abundance and size composition of the ctenophore Bolinopsis mikado were conducted in Tokyo Bay over a 5-year period from 1990 to 1994. B. mikado appeared throughout the year, and its mass occurrence was observed between August and November; annual maximum abundance ranged from 19 to 91 ind. m^–2. Water temperature seems to influence the seasonal variation of B. mikado abundance. Environmental conditions (e.g. rough waters due to a typhoon) and predation by the beroid ctenophore Beroe cucumis appear to affect annual variations of B. mikado abundance. Size frequency distributions of B. mikado indicated that its reproduction was most active in summer and fall but occurred throughout the year in Tokyo Bay. A sharp decline of the copepod population in August 1990 was probably due to predation by B. mikado which was very abundant at that time; its predatory impact was estimated to be 24 % day^–1.     

Seasonal abundance and reproductive strategy of Tetrastemma fozensis (Hoplonemertea, Nemertea) in the Villaviciosa estuary (Asturias, Northern Spain)

The seasonal occurrence and reproductive strategy of the nemertean Tetrastemma fozensis, which inhabits the mantle cavity of the bivalve Scrobicularia plana, was studied through the analysis of the temporal diversity of a T. fozensis population. Bimonthly sampling was carried out from February 2001 to January 2002 at mudflats of Villaviciosa estuary (Asturias, Northern Spain). Nemerteans were found throughout the study period, but variations in their abundance were detected, with a maximum during winter months and a minimum during summer months. Moreover, variations in mean size of nemerteans were found, with maximal sizes at the end of the spring and the beginning of the summer and minimum size at early autumn. The annual variations in population parameters (decrease in abundance and complete disappearance of large individuals in summer) suggest that T. fozensis has a distinct reproductive season and a semelparous reproductive strategy.     

规格和饥饿对刺参集群行为的影响

本文通过延时摄影技术探究了不同刺参规格（小规格、中规格和大规格）及饥饿状态（饥饿0 d、5 d、10 d和15 d）对其集群行为的影响。研究发现：不同规格刺参的聚集率和标准聚集规模之间存在显著差异（P<0.05），且呈现出随刺参规格增大而升高的趋势，表明大规格刺参更倾向于聚集，且形成的集群规模更大；除饥饿10 d和饥饿15 d的组外，各处理组之间刺参聚集率和标准聚集规模都具有显著差异（P<0.05），随着饥饿时间的延长，刺参聚集率和标准聚集规模呈先降低，后升高，再保持稳定的规律。上述结果查明了规格和饥饿对刺参集群行为的影响，可为丰富刺参行为学研究及优化刺参增养殖管理策略提供理论依据。     

Catch, survey and life-history data for shrimp (Pandalus borealis) off Jan Mayen

The Jan Mayen area has an extreme environment with low temperatures and infrequent, but abrupt temperature changes. The shrimp population here is considered to be on its edge of distribution. The life-history parameters are in the same range as in other high-latitude shrimp populations and are characterized by slow growth, large size at maturation and extended longevity. Irregular and sporadic commercial exploitation limit fishing mortality and give the population life-history parameters not previously seen in other areas. The Jan Mayen shrimp are large compared to, e.g., the Barents Sea shrimp and can reach a maximum carapace length (L_max) of 37 mm and an age of 10–11 years. The large size at sex transformation (L₅₀, >24 mm) and analyses of length–frequency distributions indicate that the shrimp may be 6–7 years of age before changing sex. The change in L_max and L₅₀ observed during the study period is probably caused by increased natural mortality due to sudden temperature changes or due to increased predation, rather than increased growth rates. The life-history strategy of shrimp in the Jan Mayen area can be explained by factors such as depth, temperature and population density variations caused by fluctuation in recruitment and mortality.The shrimp fisheries in the Jan Mayen area began in the late 1970s and reached an annual landing of 2000 tonnes in 1985, and since then landings have oscillated around 500 tonnes depending on a combination of factors. The survey indices of stock biomass varied between 3000 and 6600 tonnes. For most years, the highest shrimp densities are at a depth of 200–299 m, while large shrimp (and therefore also female shrimp) are dominant at depths greater than 300 m.Fish community data were studied as the composition of the demersal fish community is an integrated response to environmental conditions and as predation affects the shrimp stock. Polar cod and capelin are the most abundant fish species in the study area. A high number of blue whiting was registered in 1979, but the number declined in 1980 and 1981 as temperature decreased. During the surveys in 1994 and 1995, no blue whiting was registered. A few individuals were found again in the 1999 samples. The number of Greenland halibut has declined from the beginning of the 1980s to the 1990s.     

Effect of in vivo cadmium exposure on the respiratory burst of Marine Fish (Limanda limanda L.) phagocytes

Following the in vivo exposure of dab (Limanda limanda L.) to cadmium chloride, kidney phagocytes were collected and their respiratory burst measured in vitro using chemiluminescence. Fish were exposed to mean measured concentrations of 1.3, 2.7 and 5.5 mg Cd litre⁻¹ (as total cadmium ion) for a total of nine weeks, followed by a three week depuration period in clean sea water. Compared with control fish, the respiratory burst of kidney phagocytes from dab sampled after six weeks was significantly reduced in the 2.7 and 5.5 mg Cd litre⁻¹ treatments (Steel's test, p < 0.05). Significant reductions were observed in the respiratory burst of phagocytes from all cadmium exposed fish compared with control fish after nine weeks (Steel's test, p < 0.05). After a further three week depuration period in clean sea water, the respiratory burst of phagocytes from fish previously exposed to 1.3 and 2.7 mg Cd litre⁻¹ were still significantly less than in the control group (Steel's test, p < 0.05). Muscle tissue cadmium concentrations were also analysed, although there was no clear relationship between the muscle total cadmium levels and kidney phagocyte chemiluminescence. The results are discussed with respect to the possible mechanism(s) of cadmium immunotoxicity in dab and recommendations made for future work.     

盐度胁迫对香港牡蛎部分生化指标的影响

为探究盐度胁迫下香港牡蛎(Crassostrea hongkongensis)生化指标的变化规律,了解盐度适应过程中牡蛎的代谢机制,本研究以盐度0、8、16、32、40为胁迫盐度,以正常海水(盐度24)为对照,开展香港牡蛎对盐度胁迫的响应研究。结果显示,各实验盐度组糖原含量在盐度胁迫0～8 h内下降,且盐度胁迫幅度越大糖原含量降幅越大,胁迫8 h后则无明显的变化规律。腺苷酸激活的蛋白激酶(AMPK)、组蛋白/蛋白去乙酰化酶(SIRT1)和Na~+/K~+-ATP酶活力的变化规律相似:在0～8 h内,各实验组酶活力均急剧下降(SIRT1上升);8～48 h内,酶活力上升;48～120 h内,酶活力逐渐趋于平稳状态,总体表现为盐度越高,酶活力越强,并与胁迫前有明显差异,且盐度胁迫幅度越高,差异越明显。总抗氧化能力(T-AOC)总体表现出高盐胁迫下随时间的增加而升高,低盐胁迫下随时间的增加而降低,且盐度胁迫幅度越大,T-AOC活力的变化幅度越大。实验结果初步表明,香港牡蛎糖原含量与渗透压调节存在一定的关系,AMPK、SIRT1、Na~+/K~+-ATP酶活力及T-AOC均与渗透压调节密切相关,且在高盐胁迫下随时间的增加而升高,低盐胁迫下随时间的增加而降低。     

中国蛤蜊Mactra chinensis Philippi稚贝运动行为学初步研究

为从行为学角度解释中国蛤蜊(Mactra chinensis Philippi)的跑滩现象, 研究了规格、温度对其跳跃行为以及底质、流速和周期性干露对其潜沙行为的影响。结果表明, 中国蛤蜊运动能力与其规格和水温有关, 3 mm稚贝不具备跳跃能力, 5 mm以上具备跳跃能力; 跳跃频率和高度随水温的升高呈上升趋势。中国蛤蜊稚贝潜沙比例均随时间推移逐渐上升; 各时间节点中国蛤蜊稚贝在细沙底质的潜沙比例最高, 中沙底质次之, 粗沙底质最低。施加微弱水流后, 中国蛤蜊稚贝潜沙速度和比例显著增加; 流速增大到一定程度后部分稚贝被水流冲走, 潜沙比例随之下降。周期性干露会导致中国蛤蜊稚贝从底质中爬出, 干露时间越长, 爬出比例越高。干露后的稚贝重新放置于海水中, 部分稚贝会在水面漂浮一段时间后再下落至水底, 漂浮比例随干露时间的增加而增加。本研究从行为学角度初步查明了中国蛤蜊跑滩现象, 为解决中国蛤蜊防跑滩问题提供了思路。     

Feeding Habits of the Mesopelagic Fish Gonostoma gracile in the Northwestern North Pacific

The diet of Gonostoma gracile, a numerically abundant mesopelagic fish in the Subtropical Region and the Transition Domain of the northwestern North Pacific, was examined using 520 specimens collected during June–July 1988, June 1995 and November 1995. The prey included mainly copepods, ostracods, amphipods and euphausiids. Copepods and ostracods were the most abundant, comprising approximately 70% of the total diet. There was little evidence of an ontogenetic dietary shift; Pleuromamma copepods were the most abundant prey for all size classes of fish ranging from 19 to 116 mm in standard length. The size range of prey increased with growth, but all fish sizes examined fed mainly on 1–4 mm long prey. Luminescent copepods and ostracods were the most abundant prey, suggesting that G. gracile detects its prey visually. This revised version was published online in August 2006 with corrections to the Cover Date.     

大黄鱼Lcnkl3基因的分子结构、表达特征及多肽片段抗菌活性

作为重要的免疫基因, NK-lysin参与了鱼类非特异性免疫系统对抗病原感染的免疫应答过程。在之前的研究中, 我们已经发现重要经济鱼类大黄鱼(Larimichthys crocea)体内存在2种类型的NK-lysin基因。在本文中我们报道了1种新型大黄鱼NK-lysin基因Lcnkl3, 并对基因序列、表达特征及其多肽片段的抗菌活性进行了分析。Lcnkl3基因的开放阅读框长度为435 bp, 编码144个氨基酸残基, 其多肽序列Lcnkl3中包含Saposin B结构域及6个高度保守的半胱氨酸残基。系统进化分析表明Lcnkl3与大黄鱼Lcnkl2最为相似, 与其他鱼类NK-lysin多肽则差异较大。Lcnkl3基因在未受感染的大黄鱼各组织中具有不同的基础表达量, 表达水平最高的组织为心脏。在病原诱导的免疫应答过程中, Lcnkl3基因在各组织不同感染阶段的表达模式存在差异。源于Lcnkl3成熟肽序列的多肽片段对不同细菌的抑制和杀灭效果差异显著。以上结果表明Lcnkl3属于大黄鱼NK-lysin基因家族, 并参与了大黄鱼对抗病原感染的免疫过程。