Alteration of the haemocyte parameters,expression of immune‐related and neurohormone bursicon genes of giant river prawn Macrobrachium rosenbergii (de Man) in the response to salinity stress and ammonia stress

We examined the effects of salinity stress and ammonia stress on alteration of the haemocyte count, phenoloxidase activity, expressions of immune‐related genes including prophenoloxidase (proPO), crustin, heat shock protein 70 (HSP70), and expressions of stress‐responsive neurohormone (Bur‐α and Bur‐β) in the thoracic and abdominal ganglia of giant river prawn Macrobrachium. These parameters of prawn that subjected to salinity stress (transferred from 0‰ to 5‰ and 10‰), and subjected to ammonia‐nitrogen (ammonia‐N) stress (transferred from 0 to 0.262 mg/L and 0.786 mg/L) were examined after 0, 3, 6, 24, 72 and 168 hr respectively. During the initial period of 3, 6 and 24 hr, granulocyte haemocyte (granular and semi‐granular hemocyte) count and PO activity significantly decreased, while expressions of Bur‐α and Bur‐β significantly increased. After 24 hr, granulocyte haemocyte count and PO activity significantly increased, whereas expressions of Bur‐α and Bur‐β significantly decreased. The expressions of proPO, crustin and HSP70 were significantly downregulated in the prawn that subjected to salinity stress and ammonia‐N stress at all time periods of 3–168 hr. In conclusion, changes in the granulocyte haemocyte count of M. rosenbergii following salinity stress and ammonia‐N stress are closely associated with the changes of Bur‐α and Bur‐β expressions.     

Differences in uptake and killing of pathogenic and non‐pathogenic bacteria by haemocyte subpopulations of penaeid shrimp,Litopenaeus vannamei, (Boone)

Phagocytosis is an important function of both invertebrate and vertebrate blood cells. In this study, the phagocytic activity of haemocyte subpopulations of penaeid shrimp, Litopenaeus vannamei, (Boone), against pathogenic and non‐pathogenic particles was investigated in vitro. The haemocytes of penaeid shrimp were firstly separated by centrifugation on a continuous density gradient of iodixanol into four fractions with five subpopulations (sub), of which sub 1 (hyalinocytes) and sub 4 (semi‐granulocytes) have the main function in phagocytosis of both pathogenic and non‐pathogenic bacteria as well as fluorescent polystyrene beads. It was found that these haemocyte subpopulations engulfed virulent Vibrio campbellii and Vibrio harveyi at a higher rate than non‐virulent Escherichia coli and polystyrene beads. When these bacteria were mixed with shrimp haemocyte subpopulations and incubated for 180 min, the percentage of viable intracellular V. campbellii (25.5 ± 6.0%) recovered was significantly higher than the percentage recovered from V. harveyi (13.5 ± 1.1%). No viable intracellular E. coli was observed in this study. In contrast to V. harveyi and E. coli, V. campbellii containing endosomes did not acidify in time. Incubation of haemocyte subpopulations with the most virulent V. campbellii strain resulted in a significant drop in haemocyte viability (41.4 ± 6.3% in sub 1 and 30.2 ± 15.1% in sub 4) after 180 min post‐inoculation in comparison with the less virulent V. harveyi (84.1 ± 5.6% in sub 1 and 83.4 ± 4.1% in sub 4) and non‐virulent E. coli (92.7 ± 2.8% in sub 1 and 92.3 ± 5.6% in sub 4) and polystyrene beads (91.9 ± 1.6% in sub 1 and 84.4 ± 3.4% in sub 4). These findings may be a valuable tool for monitoring shrimp health and immunological studies.     

亚硝酸盐对凡纳滨对虾血细胞毒性及p53基因表达的影响

随着水产动物集约化养殖的迅猛发展，水质污染问题日益加剧，环境中亚硝酸盐的含量不断上升，成为水产养殖中诱发爆发性疾病的重要环境因子。为探讨亚硝酸盐对凡纳滨对虾（Litopenaeus vannamei）血细胞的毒性以及 p53 mRNA 的影响，通过预试验以及前期资料获取亚硝酸盐对凡纳滨对虾的毒性效应，选取0 mg／L 对照组和20 mg／L 浓度组，用流式细胞术检测血细胞活性氧（ROS）、一氧化氮（NO）含量和非特异性酯酶活性，运用荧光定量 PCR 技术检测 p53基因表达量变化。结果显示，在亚硝酸盐应激12 h 后，对虾血细胞的 NO 含量与对照组相比有显著升高（P ＜0．05），在应激24、48、72 h 后有极显著的升高（P ＜0．01）；ROS 含量在应激12、48、72 h 时有极显著升高（P ＜0．01），在应激24 h 时与对照组相比有显著的升高（P ＜0．05）。非特异性酯酶活性在应激24、48、72 h 显著下降（P ＜0．05），p53的表达水平在应激48 h 和72 h 后显著升高（P ＜0．05）。研究表明，亚硝酸盐应激诱导对虾血细胞产生了过量的 NO 和 ROS，造成氧化胁迫作用，诱导凋亡相关基因的表达，最终导致细胞凋亡。     

Effects of formalin on total haemocytes count and histopathological changes in the shrimp Litopenaeus schmitti (Pérez‐Farfante & Kensley 1997)

Formalin is used in shrimp industry to eliminate ectoparasites and as a disinfectant. However, the effect of formalin on shrimp's defense mechanism is unknown. Several biomarkers were used to assess the immune response of juveniles of the shrimp Litopenaeus schmitti exposed to different concentrations of formalin (10, 25 and 50 mg L^?1) during 24 and 48 h. Formalin concentrations of 25 and 50 mg L^?1 produced a significant reduction in the total haemocyte count at both 24 and 48 h. Peroxidase and phenoloxidase activity did not show variations. The total protein concentration in haemolymph of shrimps was not significantly different between experimental groups. Histological analysis showed hyperplasia in the gills of animals exposed at 10 mg L^?1 for 24 h; however, the exposure at 25 and 50 mg L^?1 caused necrosis in gills, hepathopancreas and the antennal gland.     

三疣梭子蟹血淋巴免疫功能的初步研究

利用API ZYM试剂盒和酶活力测试盒检测了三疣梭子蟹血细胞及血清中的19种酶类及血清中溶菌酶、碱性磷酸酶、酸性磷酸酶、超氧化物歧化酶和过氧化物酶的活性。试验结果表明,在雄蟹血清和血细胞中均检测到19种酶,在雌蟹血清和血细胞中分别检测到18种和16种。碱性磷酸酶和酸性磷酸酶的活性很低。在此基础上,研究了三疣梭子蟹血清和血细胞溶解物(HLS)对溶藻弧菌的抗菌活力,结果显示血清样品对溶藻弧菌的抗菌活力较强,但其体外作用的持续时间较短;而血细胞溶菌酶对溶藻弧菌的抗菌活力较弱。     

大弹涂鱼血细胞发生的研究

通过对大鲜涂鱼血液、肾脏、脾脏组织涂片的观察，发现其肾脏、脾脏内白细胞的发育大致经过了3个阶段，即原始阶段、幼稚阶段、成熟阶段。造血母细胞从造血器官释放入外周血中有一成熟过程。着重描述了大弹涂鱼的红细胞、嗜中性粒细胞、淋巴细胞和单核细胞发生过程中各阶段细胞的形态特征。     

Functional characterization of Farfantepenaeus californiensis, Litopenaeus vannamei and L. stylirostris haemocyte separated using density gradient centrifugation

Haemocytes from penaeid shrimp (Farfantepenaeus californiensis, Litopenaeus vannamei and L. stylirostris) were separated using a discontinuous Percoll gradient. Shrimp haemocytes were spontaneously adhered to glass, allowing slide preparations for staining and microscopic differential counting. Like other crustaceans, shrimp has three main populations differing in presence and size of cytoplasmic granules and each population seems to be biochemical or functionally compromised. Prophenoloxidase (proPO )activity was mainly located in large granules haemocytes (75%) while the small granules cells participate with 25%, but seem to be responsible for encapsulation. Haemocyte discrimination ability was tested using Sephadex? (Seph?), DEAE‐Seph? and CM‐Seph?. Only DEAE‐Seph? was encapsulated by shrimp haemocytes and provoked the release of proPO activating system, indicating the role of particle charge in the activation of shrimp immune response.     

斑节对虾血淋巴细胞对鳗弧菌的清除作用

通过检测注入斑节对虾(Penaeus monodon)血淋巴内的鳗弧菌(Vibrio anguillarumH)浓度、血淋巴细胞浓度和血淋巴细胞的组成变化,研究斑节对虾血淋巴细胞对进入体内细菌的清除作用。实验显示,斑节对虾能够迅速清除注入其体内的鳗弧菌。注射鳗弧菌悬液(107~108CFU/mL)5 min后,血淋巴中可检测到的鳗弧菌浓度是(1.4±0.6)×106CFU/mL,而2 h后所检测到的鳗弧菌的浓度仅相当于前者的3.1%。7天后,仅在部分个体的血淋巴中检测到少量鳗弧菌。伴随着血淋巴液内鳗弧菌的减少,对虾的血淋巴细胞浓度发生变化。鳗弧菌注入对虾体内5 min后,血淋巴细胞浓度为(3.51±1.69)×107/mL,注射后2 h时达到最低值,为(2.39±1.76)×107/mL,然后逐步恢复,注射后48 h达到(3.97±1.60)×107/mL。对照组血淋巴细胞浓度起初略微降低,然后逐步增加,在2 h达到最大值后又逐渐降低;除注射后48 h外,对照组血淋巴细胞浓度始终高于实验组。鳗弧菌注射入斑节对虾体内后,不同的细胞种类呈现出不同的变化规律:在注射后2 h,对照组透明细胞占总细胞数的比例显著高于实验组,对照组半颗粒细胞的含量低于实验组。注射鳗弧菌后10 min,血淋巴中颗粒细胞的相对含量显著降低,此时实验组的颗粒细胞相对含量也低于对照组。而对于降解细胞细胞核的相对含量,实验组和对照组表现出了显著不同的变化趋势。在对照组中,24 h内其含量一直在降低,而后恢复;而对照组却在注射10 min时达到最高值,而后遵循对照组类似的变化。[中国水产科学,2006,13(1):28-32]     

菲律宾蛤仔血细胞透射电镜样品制备固定液的筛选

对样品的固定是透射电镜样品制备的关键环节,通过在透射电镜下观察和比较不同固定液固定菲律宾蛤仔血细胞后血细胞超微结构的变化,初步筛选出适合菲律宾蛤仔血细胞透射电镜制样的固定液。试验结果表明,固定液可直接影响到透射电镜制样品质的高低,2.5%、5%戊二醛低渗固定液引起了血细胞细胞器的肿胀,其中线粒体肿胀尤为严重,肿胀程度2.5%戊二醛组大于5%戊二醛组;等渗固定液固定的血细胞细胞器不肿胀,但等渗5%戊二醛与4%多聚甲醛-2.5%戊二醛固定液对血细胞的精细结构保存不佳,而2%多聚甲醛-2.5%戊二醛固定液对血细胞超微结构保存较好,表现为细胞外膜结构清晰,细胞质基质丰富且分布均匀,细胞内线粒体、内质网、高尔基体、细胞核等细胞器固定效果较好。试验得出,低渗固定液会导致细胞肿胀,线粒体对渗透压的要求较其他细胞器更加敏感;提高固定液含量会减轻因低渗引起的细胞肿胀程度,但高含量固定液会损坏细胞内精细结构,故适合的样品固定液是细胞精细结构较好保存的基础。