【作者】 徐华；

【导师】 艾春香；

【作者基本信息】 厦门大学 ， 海洋生物学， 2008， 硕士

【摘要】 运用酶学分析方法和组织学技术,研究了水体中不同浓度Cd²⁺或Cr⁶⁺胁迫1d,3d,5d,7d,9d后对拟穴青蟹血细胞总数（THC）、免疫相关酶及鳃、肝胰腺显微结构等的影响,探讨Cd²⁺或Cr⁶⁺胁迫下拟穴青蟹的免疫防御机制,以期为其健康养殖水质管理提供理论指导,并为甲壳动物环境免疫学研究积累基础资料。本试验的主要结果和结论如下:1.Cd²⁺或Cr⁶⁺胁迫对拟穴青蟹血细胞总数（THC）的影响拟穴青蟹分别暴露于不同浓度Cd²⁺或Cr⁶⁺水体中1d,3d,5d,7d,9d后,其血细胞总数（THC）发生了相应的变化,且一定程度上存在时间剂量效应关系。Cd²⁺或Cr⁶⁺胁迫1d后拟穴青蟹THC即显著下降（P<0.05）,变幅与Cd²⁺或Cr⁶⁺浓度呈正相关,且拟穴青蟹对Cd²⁺的反应较Cr⁶⁺更灵敏,Cd²⁺抑制效应较Cr⁶⁺持续性更长。随着胁迫时间延长至9d后,Cr⁶⁺胁迫下拟穴青蟹THC可逐渐恢复至与对照组差异不显著的状态（P>0.05）,然而0.075mg/L、0.1 mg/L Cd²⁺组的拟穴青蟹THC仍被显著抑制（P<0.05）。2.Cd²⁺或Cr⁶⁺胁迫对拟穴青蟹血清中免疫相关酶活性的影响Cd²⁺或Cr⁶⁺胁迫对拟穴青蟹血清中免疫相关酶活性影响显著（P<0.05）。Cd²⁺或Cr⁶⁺胁迫均对拟穴青蟹血清酚氧化酶（PO）活性产生显著抑制效应（P<0.05）,但剂量效应关系不明显。Cr⁶⁺处理组的抑制效应1d后即表现出来,且持续时间较短;Cd²⁺处理组5d后才表现出来,抑制效应持续时间较长。Cd²⁺或Cr⁶⁺暴露对拟穴青蟹血清溶菌酶（U_L）活性均具有激活效应,0.075mg/L、0.1 mg/L Cd²⁺组和4.0 mg/L、8.0 mg/L Cr⁶⁺组对溶菌酶激活效应显著（P<0.05）,Cd²⁺或Cr⁶⁺对血清中溶菌酶激活效应存在时间效应关系,随着胁迫时间的延长,其激活效应逐渐减弱。Cd²⁺或Cr⁶⁺胁迫均能对拟穴青蟹血清抗菌力（U_a）产生显著抑制效应（P<0.05）,其中Cr⁶⁺的抑制效应强于Cd²⁺,并表现出时间剂量效应关系。3.Cd²⁺或Cr⁶⁺胁迫对拟穴青蟹组织、器官中免疫相关酶活性的影响Cd²⁺或Cr⁶⁺暴露对拟穴青蟹不同组织、器官中免疫相关酶类活性影响显著。Cd²⁺暴露1d后即对拟穴青蟹鳃中Ca²⁺-ATPase活性产生显著抑制效应（P<0.05）,抑制程度与Cd²⁺浓度相关性不显著（P>0.05）;Cd²⁺对拟穴青蟹鳃中Na⁺,K⁺-ATPase活性的抑制效应相对较弱,Ca²⁺-ATPase对Cd²⁺的敏感性较Na⁺,K⁺-ATPase强。拟穴青蟹鳃中Ca²⁺-ATPase、Na⁺,K⁺-ATPase活性在Cr⁶⁺暴露1d后即被显著抑制（P<0.05）,且发现随胁迫时间的延长Cd²⁺或Cr⁶⁺均可诱导拟穴青蟹鳃中Ca²⁺-ATPase、Na⁺,K⁺-ATPase活性升高。Cd²⁺暴露可诱导拟穴青蟹血清、鳃、肝胰腺、肌肉中SOD活性显著升高（P<0.05）,其中肝胰腺中SOD反应更灵敏。Cr⁶⁺暴露下拟穴青蟹血清、鳃、肝胰腺、肌肉中SOD活性均可被显著诱导升高（P<0.05）,与Cd²⁺不同的是Cr⁶⁺胁迫下鳃中SOD反应较灵敏。Cd²⁺胁迫可诱导拟穴青蟹鳃、肝胰腺、肌肉中ACP活性显著升高（P<0.05）,但Cr⁶⁺胁迫对拟穴青蟹肝胰腺、肌肉中ACP活性影响不显著（P>0.05）。Cd²⁺胁迫对拟穴青蟹鳃、肝胰腺、肌肉中AKP抑制效应显著（P<0.05）,尤其是肝胰腺、肌肉。与Cd²⁺处理不同的是,Cr⁶⁺处理能刺激拟穴青蟹鳃、肝胰腺、肌肉中AKP活性。4.Cd²⁺或Cr⁶⁺胁迫对拟穴青蟹组织、器官显微结构的影响Cd²⁺或Cr⁶⁺胁迫对拟穴青蟹组织、器官显微结构产生剂量-时间效应。胁迫9d后,其显微结构发生了不同程度的改变。0.1mg/LCd²⁺暴露9d后,拟穴青蟹鳃叶出现一定程度增厚,血腔相对增大;肝胰腺细胞界限模糊。8.0mg/LCr⁶⁺暴露下,鳃叶相对增厚,上皮层破坏;肝胰腺中血细胞增多,肝小管基膜出现断裂,界限模糊。综上所述,Cd²⁺或Cr⁶⁺胁迫对拟穴青蟹主要免疫指标影响显著,可导致机体免疫力下降。更多还原

【Abstract】 The effects of different concentrations of water-borne Cd²⁺ or Cr⁶⁺ on total haemocyte count（THC）, enzymes interrelated to immune function and histological structure of gill and hepatopancreas of mud crab Scylla paramamosain were determined by enzyme analysis and histology technique during experimental time. The aim was to clarify the mechanisms of immune defence of Scylla paramamosain under Cd²⁺ or Cr⁶⁺ stress, which will provide scientific guidelines for mud crab healthy aquaculture and pile up basic data for studying on the crustacean environmental immunology. The main results and conclusion were as follows:1. Changes of total haemocyte count of Scylla paramamosain exposed to different concentrations of water-borne Cd²⁺ or Cr⁶⁺THC of Scylla paramamosain decreased significantly when exposed to different concentrations of Cd²⁺ or Cr⁶⁺ for 1d（P<0.05）. The variation of THC was positive related to the Cd²⁺or Cr⁶⁺ concentration and more sensitive when exposed to Cd²⁺. The decreasing of THC induced by Cd²⁺（0.075mg/L, 0.1 mg/L） was significantly different from the controls after 9d exposure（P<0.05）, but Cr⁶⁺ didn’t. THC of Scylla paramamosain was approximately to control group after 9d exposed to Cr⁶⁺, however, THC still decreased after 9d exposed to Cd²⁺.2. Effects of water-borne Cd²⁺ or Cr⁶⁺ exposure on the activities of enzymes interrelated to immune function in hemolymph of Scylla paramamosainPhenoloxidase activity was significantly suppressed in hemolymph when exposed to different concentrations of Cd²⁺ for 1d（P<0.05）, exposed to different concentrations of Cr⁶⁺ for 5d. Lysozyme activity was significantly elevated in hemolymph of Scylla paramamosain when exposed to the concentrations of 0.075mg/L and 0.1 mg/L Cd²⁺, or 4.0 mg/L and 8.0 mg/L Cr⁶⁺（P<0.05）.The activation depended on exposure time of Cd²⁺ or Cr⁶⁺, which suggested a decrease in activation with prolongation of exposure time. The antibacterial activity was significantly inhibited in hemolymph of Scylla paramamosain（P<0.05）. The effect of Cr⁶⁺ exposure on antibacterial activity showed a dose-time response relationship at some degree. 3. Effects of water-borne Cd²⁺ or Cr⁶⁺ exposure on the activities of enzymes interrelated to immune function in gill, muscle and hepatopancreas of Scylla paramamosainThe Ca²⁺-ATPase activity was significantly suppressed in gills of Scylla paramamosain when exposed to different concentrations of Cd²⁺ after 1d （P<0.05）, but the changes had no relation with Cd²⁺ concentration. However, the Na⁺,K⁺-ATPaseactivity was insignificantly inhibited by Cd²⁺ compared to control group except Cd²⁺ 0.075mg/L. Na⁺,K⁺-ATPase was lower sensitive to Cd²⁺ than Ca²⁺-ATPase. The activities of Ca²⁺-ATPase and Na⁺,K⁺-ATPase were significant decreased by Cr⁶⁺ exposure in gills of Scylla paramamosain after 1d（P<0.05）. The activities of Ca²⁺-ATPase and Na⁺,K⁺-ATPase were stimulated by Cd²⁺ or Cr⁶⁺ with exposure time prolonging. The superoxide dimutase（SOD） activity were significant increased by Cd²⁺ or Cr⁶⁺ exposure in gills, hepatopancreas, muscle of Scylla paramamosain （P<0.05）. The SOD activity in hepatopancreas revealed a much higher sensitivity to Cd²⁺ than that in other tissues, whereas SOD activity in gills revealed a much higher sensitivity to Cr⁶⁺ exposure. Acid phosphatase activity in gills, hepatopancreas, muscle of Scylla paramamosain was significantly stimulated by Cd²⁺（P<0.05）, but revealed less change to Cr⁶⁺ exposure. The alkaline phosphatase activity was significant decreased by Cd²⁺ exposure in gills, hepatopancreas, muscle of Scylla paramamosain（P<0.05）, whereas Cr⁶⁺ exposure induced an increase in alkaline phosphatase activity in Scylla paramamosain.4. Effects of water-borne Cd²⁺ or Cr⁶⁺ exposure on histological microstructure of gill and hepatopancreas in Scylla paramamosainThe effect of water-borne Cd²⁺ or Cr⁶⁺ exposure on gill and hepatopancreas microstructure of Scylla paramamosain put up the dose-time effect. Some changes were observed in histolopathological microstructure of gill and hepatopancreas after 9d. The gill filament epithelium were thickened, vasal become larger compared to control group, the boundary between hepatopancreas cells were faint in Scylla paramamosain exposed to 0.lmg/L Cd²⁺ after 9d. The irregularly thickened gill lamellae epithelium disassembled were the other histolopathological effects of 8.0mg/L Cr⁶⁺ after 9d. In hepatopancreas, the basement membranes injury, the boundary between hepatic tubules was unclear, increasing hemocytes was observed.In summary, the research suggests water-borne Cd²⁺ or Cr⁶⁺ exposure caused negative effects on immune factors, led to immune disturbance in Scylla paramamosain.更多还原