Ultrastructure on oogenesis of Litopenaeus vannamei

利用透射电镜研究凡纳滨对虾卵子发生过程中细胞内部结构的变化。结果显示,卵原细胞结构简单,代谢水平低,核孔稀少,通过核周池来完成核、质之间的物质交换。卵黄发生前晚期和卵黄发生初期的卵母细胞变化显著:核膜凹凸不平,核仁数量多,核孔密集,大量核仁外排物经核孔输送到卵质中;卵质中胞器极为丰富和发达,代谢活性极强。卵黄发生旺盛期是卵黄大量形成的阶段,卵质边缘还呈辐射状排列了一圈椭圆形皮质棒,细胞出现微吞饮活动并形成卵黄膜。卵黄发生晚期卵质中充满了粗大的卵黄粒和脂滴,胞器锐减。另外,探讨了卵细胞内部结构的变化和卵黄形成的关系以及皮质棒的来源与功能。The changes of the structure in oocytes during the oogenesis of Litopenaeus vannamei were studied with transmission electron microscope. The results showed that the structure was simpler and the active metabolism is lower in oogonium.The particles from the nucleus mainly passed through the perinuclear cisternae into the cytoplasm because of fewer nuclearpores. The marked changes occurred in the latter previtellogenic oocytes and the primary vitellogenic oocytes.The nuclear changes consisted of the concavo-convex nuclear membrane,many more nucleoli, numerous nuclearpores and a large amount of nucleolus granular materials penetrating nuclear membrane into cytoplasm. There were various well-developed organelles in the cytoplasm which exhibited higher synthetic activity.The yolk granules formed mainly in active vitellogenesis. At this stage,a lap spherical cortical rods appeared radializedly at the edge of the cytoplasm.Meanwhile active microphagocytosis could be seen along the ooplasma membrane while the vitellin membrane formed on the surface of vitellogenic oocytes.The mature oocytes were full of big yolk granules and lipid droplets and the number of organelles reduced quickly. The relation between the changes of the structure in oocytes and the vitellogenesis was discussed. The origin and function of the cortical rods were also discussed.福建省教育厅自然科学基金资助项目(K02091

Effect of experimental infection with Vibrio alginolyticus on immune parameters of Pseudosciaena crocea

为了解大黄鱼在抗溶藻弧菌感染时免疫功能的变化规律,将160尾健康大黄鱼分为感染组和对照组,通过腹腔分别注射0.2mL浓度为2×107CFU.mL-1的溶藻弧菌和灭菌生理盐水,在注射0,1,3,7,11,15,19d后从两组各取6尾大黄鱼,尾静脉取血,进行外周血的血相、NBT阳性细胞数、血清抗菌活力、抗体效价等免疫学指标的测定。结果显示:在人工感染的初期,感染组大黄鱼外周血的红细胞、白细胞、淋巴细胞和NBT阳性细胞的数量及血清抗菌活力等指标均较对照组有显著提高;在注射后1d外周血粒细胞的数量显著低于对照组;抗体效价在7d开始增加,15d达到峰值,且用间接ELISA和试管凝集两种方法所得结果具有非常高的同步性。结果表明:在感染溶藻弧菌后,大黄鱼能通过红细胞和白细胞增殖、释放抗菌物质、产生特异性抗体等方式提高其对溶藻弧菌的免疫力;在感染的早期阶段非特异性免疫因子起主要作用,在感染后期阶段特异性免疫因子起重要作用;NBT阳性细胞数可以作为细菌感染的指标。To investigate the effect of experimental infection with Vibrio alginolyticus on immune parameters of Pseudosciaena crocea,one hundred and sixty healthy fish were categorized into infected and control groups.Each fish in infected group was injected intraperitoneally with 0.2 mL of bacteria suspension with a concentration of 2×107 CFU·mL-1,while each fish in control group was injected i.p.with 0.2 mL of sterile saline solution. P.crocea in both groups were sacrificed at 0,1,3,7,11,15 and 19 d post-injection to evaluate immune parameters such as haematocyte counting,NBT-positive cells,serum antibacterial activity,and serum antibody titer.The results showed that: experimental infection with V.alginolyticus would increase the numbers of erythrocytes,leucocytes,lymphocytes in peripheral blood as well as peripheral serum antibacterial activity and antibody titer of P.crocea,and reduce peripheral blood granulocytes number significantly,in comparison with those in control group.These results suggested that after infected by V.alginolyticus,P.crocea would enhance its resistance to the invaded bacteria by means such as erythrocyte and leucocyte proliferating,antibacterial substance excreting,antibodies synthesizing;non-specific immune factors including antibacterial substance in peripheral blood played an important role at the initial stages of infection,and then specific immune factors such as antibody played an important role at the later stages;the number of NBT positive cell was a good indicator of bacterial infection.国家“八六三”高新技术发展计划(2002AA639600);; 福建省自然科学基金项目(B0410022);; 福建省青年创新基金项目(2002J037

Influence of environmental factors on the adhesion of Vibrio alginolyticus to the intestinal mucus of Pseudosciaene crocea

研究了不同环境条件下溶藻弧菌对大黄鱼肠粘液的粘附作用。采用细菌计数法测定溶藻弧菌的粘附作用。粘附的细菌经SYBR GreenⅠ染色后在荧光显微镜下观察,用数码相机拍照后在电脑上计数。试验结果表明,溶藻弧菌对大黄鱼肠粘液的粘附量随菌浓度的升高而升高,并在1~1.5h内趋于饱和;粘附作用在15~30℃、pH偏酸时较强;盐度在5~35范围内对前肠粘液的粘附作用影响不明显,后肠粘液的粘附作用在此范围内随盐度增大而加强,在盐度为0时,溶藻弧菌对前、后肠粘液都无粘附作用;56℃热处理5min及60℃处理1h均能大幅减弱溶藻弧菌对两种肠粘液的粘附作用,表明溶藻弧菌表面的某些热敏结构在粘附作用中起着重要作用。根据以上结果,可以认为溶藻弧菌能够很好地粘附于大黄鱼肠粘液层,其粘附作用受温度、盐度、pH值等环境因子影响很大,溶藻弧菌表面的某些热敏结构可能在粘附过程中起着重要作用。研究结果表明,溶藻弧菌对大黄鱼肠粘液的粘附作用是可控制的,这对于鱼类养殖疾病的控制有一定的参考价值。The mucus layer is the most outer defence barrier of fish.The bacterial attachment to mucus layer is a critical step in the host colonization and subsequent occurrence of infection.Adhesion of Vibrio alginolyticus to the intestinal mucus of Pseudosciaene crocea has been investigated in order to understand the characteristics and mechanisms involved in the adherence of bacteria onto fish mucus so that preventive measures can be devised to reduce infection.In vitro adhesion assay was carried out by bacterial counting.The adhesive bacteria were stained with SYBR Green Ⅰ,observed under fluorescent microscope,counted on computer display after taking pictures.The results showed that the adhesive quantity of V.alginolyticus increases with bacterial concentrations and reached equilibrium after being incubated 1-1.5 h;the higher adhesive quantity was achieved at 15-30 ℃ and acid condition;adhesion of V.alginolyticus to the mucus of foregut was not obvious variety with salinity(5-35),but adhesive quantity to the hindgut mucus enhances while salinity increases;V.alginolyticus could not adhere to intestinal mucus without salinity;adhesive quantity reduced remarkably after heat treatment of the germ(56 ℃ 5 min,60 ℃ 1 h),which indicated that heat sensitive structure of V.alginolyticus might play an important role in adhesion.The conclusions of this paper showed V.alginolyticus could adhere to intestinal mucus of P.crocea facilely in seawater;this adhesion was remarkably affected by environmental factors,such as temperature,pH and salinity;V.alginolyticus adhered to the intestinal mucus of P.crocea by specific adhesion,some heat-sensitive structures on V.alginolyticus surface played a crucial role in adherence of the bacteria to gill mucus.The results indicated that the bacterial adhesion of V.alginolyticus to intestinal mucus of P.crocea was governable.The results obtained are useful for the epidemic control in fish culture.国家863计划项目(2002AA639600);; 福建省自然科学基金(B0410022);; 福建省青年创新基金(2002J037

Non-specific immune response of bullfrog Rana catesbeiana to intraperitoneal injection of bacterium Aeromonas hydrophila

Non-specific immune response of bullfrog Rana catesbeiana to pathogenic Aeromonas hydrophila was studied to 60 individuals in two groups. Each bullfrog in bacterium-injected group was injected intraperitoneally (i.p.) with 0.2 ml bacterial suspension at a density of 5.2 x 10(6) CFU/ml, hile each one in control group injected i.p. with 0.2 ml sterile saline solution (0.85%, w/v). Three bullfrogs in both groups were sampled at 0, 1, 3, 7, 11, 15 and 20 days post-injection (dpi) for the evaluation of non-specific immune parameters. It was observed that intraperitoneal injection of A. hydrophila significantly increased the number of leucocytes and that of NBT-positive cells in peripheral blood. Significant increases in serum bactericidal activity and serum acid phosphatase activity were also observed in the bacterium-injected frogs when compared with those in the control group. However, a significant reduction was detected in vitro in phagocytosis activity of peripheral blood phagocytes. No significant difference in changes in the number of peripheral erythrocytes, serum superoxide dismutase (SOD) activity, and lysozyme activity was detected between the two groups. It is suggested that bullfrogs may produce a series of non-specific immune reactions in response to the A. hydrophila infection.National High-Tech Research and Development Program of China [2001AA5070, 2002AA639600]; Natural Science Foundation of Fujian Province of China [B0410022, 2006F5066

Detecting macrophage migration inhibitory factor in tissues of Larimichthys crocea with Cryptocaryon irritans disease

由刺激隐核虫(CryPTOCAryOn IrrITAnS)感染引发的“白点病“为大黄鱼(lArIMICHTHyS CrOCEA)养殖业带来重大损失,但仍未找到安全、有效的治疗方法。本研究应用免疫组织化学方法,对健康大黄鱼和患刺激隐核虫病大黄鱼肠、脾、头肾与肝组织中巨噬细胞移动抑制因子(MACrOPHAgE MIgrATIOn InHIbITOry fACTOr,MIf)蛋白进行检测,研究刺激隐核虫病大黄鱼组织中MIf的表达情况及其对预后的影响。结果表明,MIf在健康大黄鱼肠、脾、头肾、肝各组织中均无表达;MIf在刺激隐核虫病大黄鱼各组织中高表达,表达强度从弱到强依次是肠、肝、脾、头肾,MIf表达阳性率分别为54%、80%、86%、90%。推测MIf在大黄鱼刺激隐核虫病发病过程中发挥作用。Cryptocaryon irritans is a holotrichous marine ciliate protozoan that causes "white spot disease", which is one of the most devastating parasites of Larimichthys crocea in Fujian Province.No safe drug strategy has been identified to prevent and cure "white spot disease".To investigate the expression and prognostic value of macrophage migration inhibitory factor(MIF) in C.irritans diseased L.crocea tissues, MIF expression was detected by immunohistochemistry of intestine, spleen, head kidney, and liver specimens of pathologically identified and healthy L.crocea tissues.None of the negative controls reacted, but MIF staining was intense in all L.crocea tissues infected with C.irritans disease.The MIF staining rates for the intestines, liver, spleen, and head kidney were 54%, 80%, 86% and 90%, respectively.The high MIF expression levels in diseased tissues indicate that MIF plays an important role in homeostasis of C.irritans infected with L.crocea.福建省自然科学基金项目(2013J01140); 2014年福建省海洋渔业资源与生态环境重点实验室科技项目(ZK2014004); 2015年集美大学福建省大学生创新项目(201510390074); 福建省研究生教育教学改革研究项目(闽教高[2015]11号;集大研[2015]19号); 厦门南方海洋研究中心科技项目; 海洋经济发展区域示范项目(14PYY050SF03;14PYY050SF03

基于小波分析的重载卡车变速箱装配品质在线检测方法研究

为确定某型变速箱装配品质，从变速箱内部结构出发，简要介绍了变速箱常见装配故障，从理论上分析了变速箱在各挡位情况下各齿轮和轴承等零件的常见故障特征频率，并搭建声压信号采集平台，采集变速箱不同挡位运转时产生的声压信号，利用小波阈值去噪法对采集的原始声压信号进行去噪，并运用小波分析对去噪后的声压信号进行分解，对相应的频段信号进行谱分析，结合理论分析与实验分析的结果，获得变速箱的装配故障特征，为检查变速箱装配品质提供依据。该研究对变速箱装配品质检测具有一定的参考意义

Detection of Vibrio fluviails in Paralichthys olivaceus using indirect fluorescent antibody technique

用灭活的河流弧菌(Vibrio fluvialis)抗原,注射免疫实验兔,制得凝集价为1∶5 120的抗血清。用试管凝集法检测了抗血清的特异性,再用免疫吸附法去除交叉反应,从而得到高效价、高特异性的抗河流弧菌血清。用所制备的抗血清在实验室中建立起河流弧菌荧光抗体检测技术(FAT),在荧光显微镜下可清楚地看到被标记的病原菌,整个检测过程只需3h。用河流弧菌感染牙鲆(Paralichthys olivaceus),24 h后,用FAT测定牙鲆的血液、肾脏和肝脏中的河流弧菌,在血液中河流弧菌的检出率最高,其次是肾脏,肝脏的检出率最低。以上结果表明:荧光抗体技术可以快速、灵敏、准确地检测出牙鲆体内的河流弧菌。Vibrio fluvialis,the pathogenic bacteria of Paralichthys oliwaceus were inactivated for vaccine.using an anti-V.fluvialis sera,agglutination titer up to 1∶5 120,was got from rabbits by injected vaccine.The serum has no cross reaction to Vibrio parahaemolyticus and other 11 trains after being absorbed.Using the anti sera,indirect fluorescent antibody technique for detection of V. fluvialis was established.The pathogenic bacteria could been observed clearly under fluorescent microscope.The whole process lasts merely 3 h.The P.oliwaceus was artificially infected by injecting of V.fluvialis,and after 24 h post injection,the blood,kidney and liver of P.oliwaceus were detected for V.fluvialis by indirect fluorescent antibody technique.The results showed that the ratio of V.fluvialis detected in blood was higher than those in kidney and liver.The results indicated that FAT would be used to detect V.fluvialis in P.olivaceus rapidly,sensitively and truly.国家863计划项目(2001AA635070);; 国家863计划项目(2002AA639600

Starvation responses of pathogenic Vibrio parahaemolyticus in natural Seawater

将副溶血弧菌培养至对数生长期,用天然海水洗脱,调整菌浓度至107个/cm3左右后置于27℃恒温培养箱进行饥饿试验.结果表明:饥饿初期细菌总数、可培养菌数和活菌数都有较大幅度上升;饥饿中、后期,总菌数与活菌数缓慢下降,而可培养菌数下降速度较快.饥饿前副溶血弧菌呈短杆状,染色均匀;饥饿后许多细胞中央出现染色较浅的区域,细胞形态变为椭圆形.副溶血弧菌对大黄鱼表皮黏液的黏附量随着饥饿时间延长而急剧下降,饥饿7 d后的黏附量接近于空白.用SDS-PAGE分析不同饥饿时期菌体蛋白质的差异,结果表明:饥饿30 d后菌体蛋白条带比饥饿前的蛋白质条带少;饥饿60 d后菌体蛋白质条带比饥饿30 d时的多,但比饥饿前少.研究结果对于揭示副溶血弧菌的流行病学特征有重要的参考价值.Pathogenic V.parahaemolyticus cells in log-phase were collected and the concentration was adjusted to 107cells/cm3 in natural sea water for starvation studies.The results showed that: total bacteria number、viable bacteria number and CFU number of V.parahaemolyticus increased remarkably at the initial starvation stages;total bacteria number、viable bacteria number and CFU number fell at metaphase and anaphase,among them,the total bacteria number and viable bacteria number fell slowly while the CFU number fell more quickly.Before starvation,the V.parahaemolyticus cells were short rods and dyed equably;after starvation,a light area appeared at the cell centre and some cells turned into oval.The amount of V.parahaemolyticus cells adhered to the large yellow croaker epidermal mucus dropped sharply during starvation,approaching the level of the control after 7 days' starvation.The cellular protein of V.parahaemolyticus was extracted and analyzed by SDS-PAGE,the results showed that: bacteria starved for 30 d had less protein bands in the gel than the unstarved ones;bacteria starved for 60 d had more protein bands compared to those starved for 30 d,but less than the unstarved ones.The results obtained were useful to the understanding of the epidemiologic characteristics of V.parahaemolyticus.国家863计划项目(2001AA635070,2002AA639600);; 福建省自然科学基金(B0410022

ENVIRONMENTAL INFLUENCE ON ADHESION OF VIBRIO ALGINOLYTICUS TO SKIN MUCUS OF PSEUDOSCIAENA CROCEA

采用3H-TdR同位素示踪方法研究了环境因子对溶藻弧菌对大黄鱼表皮粘液粘附作用的影响。试验结果表明,溶藻弧菌能很好地粘附于大黄鱼表皮粘液,其粘附量在菌浓度不超过6.52×108cfu/ml情况下随菌浓度的升高而升高;粘附量在25℃下孵育180min趋于饱和,在180min以内与孵育时间呈正相关关系;粘附作用在温度25—30℃、pH值偏酸、盐度35条件下较强;在无Na+(盐度为0)时,无粘附作用;Ca2+能显著加强溶藻弧菌的粘附作用,而Mg2+作用不明显。这些结果表明,溶藻弧菌对大黄鱼表皮粘液有较强的粘附作用,其粘附作用受温度、盐度、pH值等环境因子的影响。Fish pathogens can colonize and infect the host by attaching bacteria onto host's mucus layer. Study on the attachment and adhesion of a pathogen is important to prevent the host from infection. In this paper, we study the case of pathogen Vibrio alginolyticus and host Pseudosciaena crocea (large yellow croaker) as an example for better understanding of adhesion of the pathogen to the skin mucus of the host. The mucus samples are extracted from P. crocea with protein concentration adjusted at 1mg/ml, using microtiter plate and methyl-3H thymidine as the isotope tracer. Our results show that the quantity of V. alginolyticus adhesion increases with bacteria concentrations at the equilibrium 180min post-incubation. However, quantity does not reach an equilibrium point when the bacterial concentration ranges from 0.044 to 1.11 (OD550) 90min after incubation at 25℃; incubation temperature is one of the main factors that affect the bacterial adhesion. V. alginolyticus shows stronger adhesion in warm condition reaching the maximum at 25℃. On the other aspect, pH value affects the bacteria adhe-sion remarkably. The adhesion is remarkably stronger at pH 3---5 than at pH 7---9, peaked at pH 6. The adhesion fluctuates in seawater with different salinities optimized at 3.5%. No specific adhesion of V. alginolyticus to skin mucus is exhibited in Na+-free solution. Existence of Ca2+ but Mg2+ can obviously promotes the adhesion. All the results suggest that the pathogenic V. alginolyticus can adhere to the skin mucus of P. crocea easily in seawater; however, environmental factors of temperature, pH and salinity can influence the adhesion effectively. The optimal condition is 25-30℃, pH about 6, and 3.5% in salinity.国家863计划项目资助,2001AA635070号、2002AA639600号;; 福建省自然科学基金资助,B0410022号