中华绒螯蟹血淋巴细胞观察

本文通过显微和亚显微结构的观察研究，表明中华绒螯蟹血淋巴液中存在的细胞形态、超微结构不同的三种血淋巴细胞，即大颗粒细胞、小颗粒细胞和无颗粒细胞。血淋巴液中三种淋巴细胞的含量分别为：大颗粒细胞7.97%，小颗粒细胞15.58%，无颗粒细胞76.45%。光镜下大颗粒细胞体积最大，胞质内可见大量染色较深的粗大颗粒；小颗粒细胞体积较小，胞质内有较少的深染色细小颗粒；而无颗粒细胞体积最小，胞质内无颗粒。电镜下三种血淋巴细胞的结构特征表现为：大颗粒细胞胞质内含有大量粗大的电子致密颗粒，细胞器稀少；小颗粒细胞胞质内含数量较少、体积较小的电子致密颗粒，细胞器丰富，高尔基复合体可见；无颗粒细胞内无电子致密颗粒，细胞器稀少，部分细胞质接近透明。     

中华绒螯蟹血细胞体外吞噬能力的研究

通过体外制备血细胞单层的方法,研究了中华绒螯蟹(Eriocheir sinensis)血细胞对蜡状芽孢杆菌(Bacil-luscereus)及鼠红细胞的吞噬能力,并探讨了血清孵育对血细胞吞噬能力的影响。结果表明,以vanHARREV-ALD氏液配制的浓度为100.0mg·mL-1的L-cysteine溶液可以作为中华绒螯蟹血淋巴的抗凝剂,中华绒螯蟹血细胞在20℃下培养1h的存活率可维持在(92.5±0.9)%。中华绒螯蟹血细胞贴壁后呈扁平状,胞质可沿玻片表面迅速伸展并伸出类似于伪足的原生质突起,具有吞噬能力,对蜡状芽胞杆菌吞噬率为(5.1±1.4)%,不能吞噬鼠红细胞,血清孵育不能增强中华绒螯蟹血细胞的吞噬能力。     

中华绒螯蟹脑神经分泌细胞的研究

经光学显微镜和透射电镜观察,中华绒螯蟹的脑具有3种类型的神经分泌细胞。Ⅰ型细胞最大,胞质中存在许多大小不同的空泡,分泌颗粒数量很少;Ⅱ型细胞中等大小,细胞器发达,分泌颗粒数量较多,形态多样;Ⅲ型细胞最小,分泌颗粒数量最多,细胞器很少。     

中华绒螯蟹血细胞数及离体后形态学变化

中华绒螯蟹循环血液中血细胞总数为(13357±7196) cells·mm^-3,其中大颗粒细胞所占(49.8±7.5)%,其长轴(16.6±1.1) μm,短轴(10.2±0.6) μm;大小颗粒中间型细胞所占比例(20.6±5.3)%,其长轴(13.1±1.2) μm,短轴(8.4±0.5) μm;小颗粒细 胞所占比例(29.3±4.8)%,其长轴(12.4±1.2) μm,短轴(8.1±0.4) μm;无颗粒细胞所占比例(0.2±0.2)%,其长轴(11.3±1.3) μm,短轴(8.9±1.1) μm。血液离体后,在血液完全凝固的(97.7±25.3) s时间内,只有无颗粒细胞和小颗粒细胞形态结构发生变化,细胞及其胞核立即膨大,核质比迅速增大,细胞膜破损崩解,胞浆溢出,血液完全凝固后这些细胞形态结构继续变化,主要表现为胞质内线粒体、内质网等细胞器扩张溶解消失,小颗粒细胞中的小颗粒内含物渗出或溢出,颗粒空泡化或崩解,细胞核固缩等变化过程 。而大小颗粒中间型细胞和大颗粒细胞形态结构的变化明显慢于前两种血细胞,在细胞离体后5 min时,细胞和细胞核才开始出现膨大,并逐渐出现细胞膜、线粒体、内质网溶解消失,颗粒空泡化或崩解以及细胞核固缩等形态结构的变化。从血液离体后血细胞形态结构随时间变化的特点,推测无颗粒细胞和小颗粒细胞可能类似于脊椎动物的血小板,通过释放胞内凝血物质参与血凝的级联反应。     

中华绒螯蟹的血细胞组成、分类及免疫学功能

为了解中华绒螯蟹血细胞组成、分类及其在免疫应答过程中起到的重要作用,本研究通过细胞化学和细胞酶学分析,并结合细胞形态观察,对中华绒螯蟹血细胞的组成、分类进行了研究。同时,通过人工感染嗜水气单胞菌后血液的总血细胞数(THC)和不同类型血细胞数(DHC)的变化,研究不同类型血细胞在免疫应答过程中所发挥的作用。结果显示,依据本实验分类方法,中华绒螯蟹血细胞可以分为4类:大颗粒细胞(G)、中间型颗粒细胞(IG)、小颗粒细胞(SG)和透明细胞(H)。其中小颗粒细胞数量最多,约占33.54%±0.98%,中间型颗粒细胞最少,仅占15.31%±2.01%。4种类型细胞中均含有多糖成分而不含脂质,只有大颗粒细胞在酸性磷酸酶、碱性磷酸酶、β-葡萄糖醛酸酶以及酚氧化酶染色中发现阳性反应。此外,嗜水气单胞菌感染后中华绒螯蟹总血细胞数在3 h后明显升高,在6 h达到峰值,约9.57×106个/m L,并显著高于未处理组和生理组;在感染过程中,大颗粒细胞数量明显下降而透明细胞数量明显上升,这种现象在6 h时达到顶峰并随着时间延长而逐渐恢复至正常水平。研究表明,在中华绒螯蟹非特异性免疫应答过程中,透明细胞主要通过大量增殖执行吞噬功能来参与免疫应答,而大颗粒细胞主要通过裂解释放胞质中所含免疫相关酶参与免疫反应。其中,各类型细胞之间可能存在相互转化作用,而中间型颗粒细胞为这种转化中的过渡类型。     

图像流式细胞仪在中华绒螯蟹血细胞分群及吞噬功能研究中的应用

为了更精确地对甲壳动物血细胞进行快速分类和功能分析,本研究以中华绒螯蟹为例,依据其血细胞内具有颗粒结构的特征,与常规显微观察对比,探讨了一种基于图像流式细胞仪的血细胞自动化分类新方法,并测量了体质量为(10±3) g的中华绒螯蟹的血细胞对直径1μm微球的吞噬情况。结果显示,两种方法都可将中华绒螯蟹血细胞分为4个类群。显微镜观察分类基于胞内可见颗粒,但由于缺乏精细的量化标准,批次样品的人工辨识结果波动较大;而图像流式方法以胞内全部颗粒结构为对象,利用高精度检测模块测量,结果更准确、客观,而且通量高、重复性好。测量结果显示,中华绒螯蟹血淋巴中无颗粒、小颗粒、中颗粒及大颗粒细胞占比分别为40.62%±2.65%、36.68%±6.84%、7.80%±1.16%和16.51%±5.60%,依据测量的颗粒特征区分的4个类群界限清晰,缺乏过渡样点,提示各类细胞之间可能没有相互转化。进一步的活体微球吞噬实验证实,中华绒螯蟹的4类血细胞都具有吞噬功能,并以无颗粒细胞为主要吞噬类群;吞噬微球的细胞比例在注射后6 h内呈钟型曲线变化,4 h可达峰值(5.69%±0.44%),表明中华绒螯蟹血细胞能高效清除血淋巴中的异物。研究表明,图像流式细胞仪适合于中华绒螯蟹的血细胞分类分析和功能研究,本研究结果为同类研究提供了重要参考,将有助于更全面了解甲壳动物血细胞的功能。     

驼背鲈血细胞的超微结构

利用电镜技术对驼背鲈（Cromileptes altivelis）外周血细胞的超微结构进行了研究。结果表明,电镜下可区分6种类型的细胞：红细胞、粒细胞、单核细胞、淋巴细胞、血栓细胞和浆细胞。红细胞呈圆形或椭圆形,可见线粒体、少数囊泡;根据细胞中的颗粒形态大小和细胞核的形态,可将粒细胞分为4种类型：I型粒细胞、II型粒细胞、III型粒细胞和IV型粒细胞。单核细胞圆形,表面较平整,偶有伪足伸出,空泡多见;淋巴细胞的胞质少,仅在细胞核边缘处形成薄薄的一层,细胞器少;血栓细胞异染色质丰富,沿核膜呈带状分布,胞质中有较多大小不等的空泡和少量的线粒体;浆细胞中粗面内质网成层分布包绕在核周围。此外可见到嗜曙红细胞吞噬红细胞、血栓细胞成群聚集分布的现象。     

中华绒螯蟹Y-器官在蜕皮周期中的超微结构

以光镜和电镜观察处于蜕皮周期不同阶段的中华绒螯蟹(Eriocheirsinensis),结果显示,中华绒螯蟹具Y-器官(YO)1对,卵圆形,直径1 5～2 0mm,苍黄色,位于头胸部鳃腔前端,大颚外侧内收肌腹缘,邻近头胸甲内侧上皮。YO细胞排列成索状,细胞索之间为血窦,YO的细胞索由一种细胞构成,细胞核质比高。YO细胞具有类似脊椎动物合成类固醇激素的典型超微结构特征,蜕皮前期YO细胞的光面内质网、管嵴状线粒体和游离核糖体丰富;蜕皮后期和蜕皮间期的内质网、线粒体和游离核糖体明显减少;青春蜕皮后的YO出现大面积细胞凋亡,YO趋向退化。     

中华绒螯蟹围食膜结构观察及其主要蛋白质种类的鉴定

为揭示中华绒螯蟹围食膜的分泌、形态和功能,实验采用组织切片技术和电镜技术观察了中华绒螯蟹消化道及围食膜的基本形态,采用几丁质酶溶解法和SDS-PAGE电泳法研究了围食膜的基本组成成分,采用液相色谱电离串联质谱(LC-ESI-MS/MS)分析了围食膜蛋白的种类。显微切片结果显示,中华绒螯蟹整个消化道表面都有围食膜存在,除中肠外围食膜区室化作用明显;围食膜与上皮细胞分离时,柱状细胞变成不规则圆形,核消失。电镜结果显示,围食膜形成时上皮细胞内含有大量线粒体和分泌泡;中肠上皮细胞表面密生微绒毛。围食膜能被几丁质酶溶解,围食膜蛋白质SDS-PAGE图谱显示有7条比较清晰的电泳条带,蛋白质分子量主要分布在40 ku以上。LC-ESI-MS/MS分析结果发现,中华绒螯蟹围食膜蛋白主要包括钠、钾-ATP酶、ATP合成酶、肌动蛋白、微管蛋白、硫氧还原蛋白、加氧酶等。研究表明,中华绒螯蟹的围食膜由上皮细胞分泌形成,可能参与免疫、渗透调节和营养物质转运等生理过程。     

中国大陆沿海六水系绒螯蟹_中华绒_省略__群体亲缘关系_生化遗传差异分析

采用聚丙烯酰胺凝胶电泳分析比较了分布于我国大陆沿海 6个主要水系的两种绒螯蟹 (中华绒螯蟹、日本绒螯蟹 )群体间的生化遗传差异。结果发现 ,(1)辽河、黄河、长江和瓯江中华绒螯蟹群体间的生化遗传差异不显著 ,四个群体间的遗传距离D =0 .0 0 0 6～ 0 .0 0 53,属种内群体间差异。 (2 )珠江和南流江日本绒螯蟹群体间的生化遗传差异也不显著 ,两个群体间遗传距离D=0 .0 0 0 7,尚未达到亚种分化水平。 (3)辽河、黄河、长江和瓯江中华绒螯蟹与珠江和南流江日本绒螯蟹在SOD同工酶表型上有明显差异 ,SOD - 2位点仅在辽河、黄河、长江和瓯江中华绒螯蟹中表达 ,而SOD - 4位点仅在珠江和南流江日本绒螯蟹中表达 ,这些可作为中华绒螯蟹与日本绒螯蟹群体区分的生化遗传标志。辽河、黄河、长江和瓯江中华绒螯蟹群体同珠江和南流江日本绒螯蟹群体间的遗传距离较大 ,D =0 .0 72 1～ 0 .0 74 7。 (4 )聚类分析表明 ,在我国大陆沿海水系分布的绒螯蟹可分为两大类群 ,辽河、黄河、长江和瓯江水系的绒螯蟹属北方类群 ,即中华绒螯蟹 ;珠江和南流江的绒螯蟹属南方类群 ,即日本绒螯蟹。     

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.