草鱼鳃组织蛋白双向电泳技术的建立及优化

为建立并优化草鱼鳃蛋白质组双向电泳的技术体系,实验将草鱼鳃经裂解处理后,通过固相p H梯度胶条进行一向等电聚焦和SDS聚丙烯酰胺凝胶垂直电泳进行蛋白分离,对不同的裂解液配方、蛋白纯化方法、IPG胶条的分离范围、上样量和染色方法等进行了探索和优化,并应用lmage Master 2D Platinum 7.0软件对电泳图谱进行了分析。结果表明:采用裂解液中添加DTT来取代Tris和TBP、选择分离范围为pH4-7的IPG胶条和150μg的主动水化上样,电泳结束后对凝胶进行硝酸银染色,获得了覆盖范围广、低背景、高分辨率、适合差异蛋白分析的双向电泳参考图谱。     

Early stages of Margaritifera margaritifera glochidiosis in Atlantic salmon: Morphopathological characterization

Freshwater mussels of the order Unionida encyst into the fish mucosa to metamorphose and complete their life cycle, causing a parasitic disease known as glochidiosis. This parasitic stage represents a bottleneck for the survival of naiads, particularly for critically endangered species as Margaritifera margaritifera; however, little is known about the events occurring during this critical stage. Therefore, this study aimed to histologically characterize the development of M. margaritifera glochidiosis in Atlantic salmon to get insight into the pathogenesis of this interaction. Fish exposed to glochidia were sampled during the first 44 days post-exposure, and organs were observed by stereomicroscopy and light microscopy. Glochidia attached to the gills by pinching the lamellar epithelium, whereupon an acute proliferative branchitis engulfed most of the larvae. However, during the first 14 days, a severe detachment of unviable glochidia occurred, associated with the presence of pleomorphic inflammatory infiltrate and epithelial degeneration. In the cases where larvae remained attached, a chronification of the lesions with none to scarce inflammation was observed. These results provide key information to better understand the complex host–parasite interaction during the early stages of glochidiosis and provide valuable information to optimize artificial rearing of naiads in conservation of threatened freshwater mussel populations.     

Methacarn preserves mucus integrity and improves visualization of amoebae in gills of Atlantic salmon (Salmo salar L.)

Two aqueous fixation methods (modified Davidson's solution and modified Davidson's solution with 2% (w/v) Alcian blue) were compared against two non‐aqueous fixation methods (methacarn solution and methacarn solution with 2% (w/v) Alcian blue) along with the standard buffered formalin fixation method to (a) improve preservation of the mucous coat on Atlantic salmon, Salmo salar L., gills and (b) to examine the interaction between the amoebae and mucus on the gill during an infection with amoebic gill disease. Aqueous fixatives demonstrated excellent cytological preservation but failed to deliver the preservation of the mucus when compared to the non‐aqueous‐based fixatives; qualitative and semi‐quantitative analysis revealed a greater preservation of the gill mucus using the non‐aqueous methacarn solution. A combination of this fixation method and an Alcian blue/Periodic acid–Schiff staining was tested in gills of Atlantic salmon infected with amoebic gill disease; lectin labelling was also used to confirm the mucus preservation in the methacarn‐fixed tissue. Amoebae were observed closely associated with the mucus demonstrating that the techniques employed for preservation of the mucous coat can indeed avoid the loss of potential mucus‐embedded parasites, thus providing a better understanding of the relationship between the mucus and parasite.     

盐度胁迫对三疣梭子蟹鳃Na+/K+-ATPase酶活的影响

通过钼蓝法测定三疣梭子蟹在3组实验盐度的胁迫过程中第2对和第6对鳃Na⁺/K⁺-ATPase酶活的变化,比较了3组实验盐度胁迫1 d时,鳃Na⁺/K⁺-ATPase的酶活大小。结果表明,在盐度胁迫初期,3组实验盐度下第2对和第6对鳃Na⁺/K⁺-ATPase的酶活下降;之后,各组实验盐度下第2对和第6对鳃Na⁺/K⁺-ATPase的酶活开始随胁迫时间增长而上升;最后,各组实验盐度下第2和第6对鳃Na⁺/K⁺-ATPase的酶活下降并趋于稳定。另外,胁迫1 d时,各组实验盐度下三疣梭子蟹前5对鳃Na⁺/K⁺-ATPase的酶活显著低于后3对鳃Na⁺/K⁺-ATPase的酶活。三疣梭子蟹对盐度变化的调节可分为被动应激期(酶活力下降)、主动调节期(酶活力逐渐上升)和适应期(酶活力稳定);三疣梭子蟹后3对鳃是离子转运、渗透压调节的主要部位。     

Analysis of mesh breaking loads in cotton gill nets: Possible solution to ghost fishing

ABSTRACT:   A small number of fishers in Chiba Prefecture of eastern Japan use cotton gill nets to catch Japanese spiny lobster Panulirus japonicus. To examine the advantages of cotton gill nets, we analyzed changes in mesh breaking load of a new cotton gill net used in a fishing operation. A new cotton gill net was also soaked in a seawater tank to simulate ghost fishing conditions. The average mesh breaking load of new cotton mesh was 50.3 N. This value decreased to 19.0 N after 38 days (∼912 h), and after 82 days (∼1968 h) the mesh could be easily torn (breaking load 0.07 N). Under fishing conditions, the cumulative soak time was only 744.4 h over 19 months. The average breaking load at the end of this period was 43.1 N, a strength 86% that of the presoaked mesh. The mesh breaking load of a cotton gill net continuously soaked for 744.4 h was 26.1 N, as estimated from tank experiment data. Thus, a cotton gill net maintains reasonable strength under typical use conditions, but will degrade if lost at sea.     

鳙鳃凝集素的分离纯化及理化性质的研究

利用DEAE-Sepharose FF离子交换和Sephacryl S-200 HR及Superdex 200 10/300 GL分子筛层析技术,从鳙鳃组织中分离纯化到一种岩藻糖专一的凝集素,命名为GANL.在还原SDS-PAGE电泳上显示单一蛋白染色带,其亚基相对分子质量为37 kD.经Superdex 200凝胶过滤层析测得其天然相对分子质量为220 kD.GANL的中性糖含量为13.4%.因此,GANL作为一种由相同亚基组成多亚基糖蛋白.GANL对兔红细胞有专一的凝集活性,其凝集活性不依赖Ca~(2+).在被测的单糖、双糖及糖蛋白中,仅岩藻糖能抑制其凝集活性.氨基酸分析表明GANL的Asp、Glu、Leu、Val、Lys的含量较高,Cys-S的含量为0.81%.GANL的最适pH为8～9;具有很高的热稳定性,最适温度为50 ℃.     

浅色黄姑鱼鳃结构及其呼吸面积的研究

应用扫描电镜技术对体重范围在4.75～35.51g的浅色黄姑鱼（Nibea coibor）鳃结构及鳃小片进行了观察与呼吸面积计算,研究体重、体长与呼吸面积之间的相关关系。结果表明,浅色黄姑鱼的平均相对呼吸面积为4.01±1.62cm^2·g^-1。体重与一侧鳃丝总数的相关性N=28.81＋158.45lnW（R^2=0.9670,P〈0.01）较体长与一侧鳃丝总数的相关性N=17.31L^1.4542（R^2=0.8241,P〈0.01）更为显著。单位mm鳃小片的数目随着体重的不断增加显著减少,N=49.971W^-0.3909（P〈0.01）。单个鳃小片的面积随体重的增大而显著增加,a=0.3199W^0.2528（P〈0.01）。总呼吸面积随体重的增大而显著增加,A=11.559W^0.7109（P〈0.01）。相对呼吸面积随体重的增大而显著减小,RA=11.561W^-0.2904（P〈0.01）。     

盐度突变对凡纳滨对虾组织碳酸酐酶活性的影响

采用改进的△pH法检测凡纳滨对虾组织碳酸酐酶活性,监测了环境盐度从5突变到25后对虾鳃和触角腺组织碳酸酐酶活性及其血淋巴渗透浓度的动态变化。结果表明:(1)鳃组织碳酸酐酶活性在盐度突变后4 d内没有显著变化,活性水平在(21.68±1.25)μmol CO2/(mg.min),第5~11天有显著性的增高现象,在第10天活性最高,为(43.03±2.82)μmol CO2/(mg.min),第12天回落至起始水平;(2)触角腺碳酸酐酶活性动态变化的模式与鳃相似,起始增高时间比鳃晚6 d;(3)触角腺碳酸酐酶活性显著增高起始的时间与鳃组织碳酸酐酶活性开始显著下降的时间有关联性;(4)在低盐度时,凡纳滨对虾属于强高渗调变生物。     

氯氰菊酯对鲤鱼亚急性毒性研究

采用室内饲养方法,研究氯氰菊酯杀虫剂对鲤鱼的亚急性毒性效应。鲤鱼幼鱼经急性毒性试验和0、0.28、0.58、1.14μg·L-1氯氰菊酯暴露35d后,测定生长指标并对鳃组织病理切片进行观察。结果表明,氯氰菊酯对鲤鱼幼鱼的96hLC50为12.6μg·L-1;在1.14μg·L-1浓度组,可以明显地观察到氯氰菊酯对鲤鱼生长的抑制作用,在0.58和1.14μg·L-1浓度组,可以观察到鳃组织有明显损伤。因此氯氰菊酯对鲤鱼的最大允许浓度为0.28～0.58μg·L-1。