GeO2对海带幼孢子体培育过程中硅藻污染的抑制效应

采用叶绿素荧光技术,以光系统Ⅱ最大荧光产量(Fv/Fm)为指标,结合形态显微观察,研究了GeO2在海带幼孢子体培育过程中菱形藻污染的抑制效应。结果表明,1)对于菱形藻,当GeO2浓度在5mg/L以上时,对其Fv/Fm值有显著影响,达到或超过15mg/L时菱形藻趋于死亡。2)对于海带幼孢子体,GeO2浓度在20mg/L以上时,对其Fv/Fm值有显著影响,达到或超过30mg/L时其趋于死亡。3)对于受到菱形藻污染的海带幼孢子体,GeO2浓度在15mg/L以上时对其Fv/Fm值有显著影响,达到或超过20mg/L时其趋于死亡。综合来看,当GeO2浓度为5～15mg/L时,菱形藻光合作用受到较强的抑制,而对海带幼孢子体影响不大。     

拟态弧菌的绿色荧光蛋白标记及其在感染草鱼体内的动态分布

为了示踪研究拟态弧菌感染草鱼的动态过程,将增强型绿色荧光蛋白编码基因EGFP克隆至质粒pBAD24,并转化到拟态弧菌04-14菌株构建荧光标记重组菌.重组菌经阿拉伯糖诱导后,能高效表达EGFP蛋白;荧光显微镜观察和流式细胞仪检测均发现重组菌能够发出明显的绿色荧光信号,且传至30代后质粒稳定率仍为100％;生物学特性检测结果显示,与野生株相比,重组菌的形态、生长特性和细胞黏附性均未发生明显改变.用标记重组菌浸泡感染草鱼,定点采集鳃、肠道、肌肉、头肾、脾脏和肝脏,借助荧光信号检测4d内细菌在不同组织脏器中的动态分布.结果发现感染4h后即可在肠道和鳃中检测到绿色荧光信号,标记菌检出量分别为3.60×108和2.36×106 CFU/g,直至10 h,其含量无明显变化,12 h后含菌量逐渐下降,但持续存在直至鱼死亡.标记菌在肌肉、头肾、脾脏和肝脏中呈现相似的动力学,感染24 h后才检测到荧光信号,24～ 85 h时间段含菌量呈现先增加后下降的变化,48 h达到峰值,检出量分别为9.58×104(肌肉)、8.75×104(头肾)、1.50×104(脾脏)和4.50×104 CFU/g(肝脏),但均低于肠道中的检出量,结果表明肠道是拟态弧菌黏附定植与繁殖的主要靶器官.     

海带孢子体光合活性对不同温度和光照的响应

为了从生理生态学角度解答温度和光照对海带孢子体生长过程的影响,探索海带孢子体对温度和光环境的生理响应机制,实验在测定养殖海域海带孢子体生长参数的基础上,设定了6、10、14和18°C 4个水温梯度的海带孢子体暂养实验,以及它们在0、25、70、133、230、317、421、582、786μmol photons/(m2·s) 9个光合有效辐射(PAR)梯度下的光合活性荧光参数测定。结果显示:①在6°C水温条件下,海带孢子体荧光参数Fv/Fm和Fv/F0最大,分别为0.71和2.40;在18°C水温条件下,其Fv/Fm和Fv/F0最小,分别为0.65和1.85;②暂养海带孢子体的光化学淬灭(qP)和非光化学淬灭(qN或NPQ)在18°C水温条件下达到最大,分别为0.92和3.29;③海带孢子体的快速光曲线随着光合有效辐射(PAR)的增强呈现先上升后下降的趋势;④海域养殖海带孢子体的最大叶长增长速率、叶宽增长速率和干重增重率分别为1.34 cm/d、0.33 cm/d和1.01 g/d。研究表明,海带孢子体干重生长率变化与不同水温条件下的快速光曲线变化一致,高温抑制了海带孢子体光合效率;...     

绿色荧光蛋白基因重组子的构建及其在鱼类受精卵中的表达

通过体外重组，将绿色荧光蛋白基因编码序列克隆到鲤鱼肌动蛋白基因启动子下游，构建成能在鱼体内表达绿色荧光蛋白的重组分子。用限制性内切酶PvuI酶切线性化后，通过显微注射法导入金鱼受精卵内，在转化后48h，可观察到绿色荧光，经PCR初步筛选后，对显阳性个体进行Southern杂交检测，结果表明，转化个体表现出较强的杂交信号。这说明绿色荧光蛋白基因能在金鱼体内整合、表达。     

温度和时间对海带(Saccharina japonica)孢子囊孢子放散及后期生长发育的影响

为了能够节约制冷成本,合理延长种海带在海上的预留时间,系统研究了不同温度(20、21、22、23、24℃)和培养时间(4、8、12、16d)对两个海带品系(“205”和“C033”)孢子放散能力及胚孢子萌发率、受精率、雌配子体发育和幼孢子体生长的影响.结果显示,当海水温度达到24℃时,孢子囊几乎丧失了放散能力,20-23℃的游孢子放散量差别不显著.在相同温度下,孢子囊培养时间为12、16d的胚孢子萌发率大于4、8d;同时,孢子囊培养16d的雌配子体和幼孢子体具有最小值.在相同培养时间下,23℃(或24℃)的雌配子体和幼孢子体远远小于20-22℃的.相关性分析表明,海带雌配子体大小与幼孢子体大小具有显著正相关性(P＜0.01).结果显示,当温度达到23℃或培养时间达到16d时,海带孢子囊受到了胁迫,不利于雌配子体的发育和幼孢子体的生长.温度对孢子囊的作用更迅速,而时间对孢子囊的作用较为缓慢,可能是一种累积效应.     

海带染色体的DAPI染色及核型初步分析

鉴于海带染色体比较小且数目存在分歧等原因,利用0.2%秋水仙素对海带配子体及孢子体处理10 h左右,经过卡诺试剂固定、多种酶组合处理及30 cm的高位滴片,可以获得质量比较高的海带染色体;使用灵敏度高、特异性强的DNA荧光染料DAPI进行染色,结果显示,海带雌、雄配子体的染色体各为31条,孢子体染色体为62条,大多为短杆状或者点状;雌配子体染色体的大小为0.78～2.61μm,稍大于雄配子体(大小为0.57～2.17μm)。根据染色体的大小,对海带配子体的染色体核型进行了初步分析。这些结果为分子标记的染色体定位等细胞学研究奠定了技术基础。     

利用液相氧电极技术研究“海天1号”海带(Saccharina japonica)幼孢子体光合及呼吸速率

“海天1号”海带是南方耐高温品种与北方高产品种杂交后,经过几代选育获得的海带新品系,具有产量高、耐高温、成熟稍早等特点(暂命名为“海天l号”海带).为探究“海天1号”海带幼孢子体在不同环境因子下的生理适应性,设置了温度、光强及营养盐3个单因素实验,利用液相氧电极技术研究其对“海天l号”海带幼孢子体(1-2 cm)表观光合速率(Pn)和呼吸耗氧速率(R)的影响,并测定相对生长速率(Relative growth rate,RGR).结果显示:(1)温度为14℃、光强为80 μmolphotons/(m2.s)、氮营养盐为6 mg/L(N/P=10∶1,下同)环境下,幼孢子体的RGR和Pn均较大.在温度为22℃、光强为1 00 μmol photons/(m2·s)、氮营养盐浓度为10 mg/L条件下,R最强.(2) 22℃组的幼孢子体生长受到抑制,其RGR显著低于14℃组(P＜0.05).20 μmol photons/(m2·s)与80 μmolphotons/(m2·s)光强组的RGR存在显著性差异(P＜0.05).单因素方差分析显示,5个营养盐浓度组之间幼孢子体的RGR无显著性差异(P＞0.05).(3)随着温度、光强、营养盐浓度增加,幼孢子体的Pn呈先上升后下降的趋势,14℃组Pn显著高于其他温度组,氮营养盐浓度4 mg/L和6 mg/L组的Pn显著高于2、8和10 mg/L组(P＜0.05).在本实验条件下,R和Pn呈负相关.(4)光强对幼孢子体P/R(总光合速率/呼吸耗氧速率)的影响较小,温度和营养盐浓度对其影响较大.正交实验结果显示,适宜“海天1号”海带幼孢子体培养的条件为光强50 μmol photons/(m2·s)、温度16℃、氮营养盐浓度5 mg/L.     

镉对凡纳滨对虾离体血细胞的毒性影响

对凡纳滨对虾血细胞进行离体镉离子应激,Cd2+浓度为10-3-10-9M,在应激6h后取样,利用流式细胞术（FCM）检测血细胞的活性和活性氧（ROS）含量。结果显示,当Cd2+浓度为10-5M时,血细胞的ROS含量显著升高;当Cd2+浓度为10-3和10-4M时,血细胞的死亡率和ROS含量均显著上升。这些结果表明Cd2+的细胞毒性作用与其浓度相关,Cd2+浓度达到10-5M时即可诱导血细胞ROS的产生,但当Cd2+浓度达到10-4M及以上时,被诱导产生的ROS对细胞造成氧化伤害从而导致细胞死亡。     

高温对海带(Saccharina japonica)抗氧化酶及叶绿素荧光参数的影响

以海带(Saccharina japonica)幼苗为材料,研究了高温(25℃)条件下其抗氧化酶、叶绿素含量、叶绿素荧光参数的变化.研究表明,(1)高温组的可溶性蛋白含量在处理12 h后达最大值(P＜0.05),相比初始含量升高了37.9％.(2)随着胁迫时间的延长,高温组的丙二醛(MDA)含量呈增加趋势,在48h时达最大值,与对照组差异显著(P＜0.05).(3)高温组海带幼苗的叶绿素含量随胁迫时间延长呈下降趋势,胁迫48 h时,叶绿素含量达最小值,与初始值相比下降了38.5％ (P＜0.05).(4)在高温条件下,过氧化物酶(POD)活性随胁迫时间延长逐渐下降,在48 h时达最小值,与初始值相比下降47.2％,呈显著性差异(P＜0.05);超氧化物歧化酶(SOD)活性呈现波动趋势,处理8h后,SOD活性达最小值;40h后,SOD活性达最大值,与初始值相比上升了36％ (P＜0.05).过氧化氢酶(CAT)活性随胁迫时间延长呈下降趋势,处理36h后,海带幼苗的CAT活性略有升高;处理48 h后,CAT活性达最小值,与初始值相比下降了75.8％ (P＜0.05).(5)高温条件下,海带幼苗的光化学最大量子产量(Fv/Fm)随着胁迫时间延长呈现下降趋势,处理48 h后,高温组的Fv/Fm达最小值,与初始条件相比下降了56.8％,呈显著性差异(P＜0.05).本研究可为海带的耐高温品种选育提供理论依据.     

9个海带栽培品种自交系后代遗传多样性和亲缘关系的AFLP分析

于2007-2008年采集养殖生产中使用的海带烟杂和单克隆海带配子体杂交培育的东方3号等9个海带(Laminaria japonica)品种/系成熟孢子体,采集游孢子后,通过人工育苗获得自交子一代.以青岛野生海带自交子一代作为对照,运用AFLP分子标记技术对以上10种海带幼孢子体进行遗传多样性和亲缘关系分析.用10对引物组合共扩增条带434条,其中多态性带141条,多态性平均检出比例32.5%.UPGMA聚类分析,可将10个海带品种/系自交子一代分成三大类群(相似系数0.87).其中90%受试海带品种/系亲本的纯合性较高,自交系个体后代遗传相似系数在0.97以上.分析结果表明,中国北方栽培海带品种/系与青岛的野生海带可能来源于同一种群,而福建的栽培海带则在亚热带水域长期栽培后特化成一个相对独立的地理种群.早厚成海带和真海带之间有较近的亲缘关系,推测早厚成海带的亲本可能是真海带.而烟杂海带亲本杂合度明显高于其他海带品种/系,从品种的角度考虑,还需进一步筛选纯化.     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.