【作者】 张灵燕；

【导师】 张秀梅；

【作者基本信息】 中国海洋大学 ， 增殖养殖工程， 2010， 硕士

【摘要】 褐牙鲆(Paralichthys olivaceus)是分布于我国、日本和韩国等东北亚沿岸的底栖鱼类,具有重要的经济价值,在海水鱼类养殖业中占有重要地位。本实验以褐牙鲆幼鱼为研究对象,研究了钙镁离子总浓度、钙离子浓度及盐度驯化对其血清渗透压的影响,并对其渗透调节机制进行了探讨,主要得到以下结果及结论：1、钙离子浓度对褐牙鲆幼鱼血清渗透压的影响及其渗透调节机制的研究Ca2+由自然海水(9.7 mM)向低浓度组(a1：1.25 mM)突变216 h内,褐牙鲆幼鱼血清渗透压在6 h时显著升高(P<0.05),12 h后即恢复至正常水平；鳃和肠Na--K+-ATPase和Ca2+-ATPase活力略有下降,呈现下降(6 h和72 h)后回升(12 h和96 h)并趋于稳定的趋势,肾Na+-K+-ATPase和Ca2+-ATPase活力显著升高(P<0.05),呈现上升(6 h)后下降(24 h)并趋于稳定的趋势；血清Na+、Cl-和K+含量无显著变化,实验6 h后,血清Ca2+含量下降并维持稳定,但与对照组无显著差异(P>0.05),血清Mg2+含量则显著上升并维持稳定(P<0.05)；光镜结果显示(216 h),鳃丝横切面内氯细胞密度以及大小与对照组均无显著差异(P>0.05)；电镜结果显示(6 h),氯细胞微管系统分支减少。Ca2+由自然海水(9.7 mM)向高浓度组(a3:37.5,a4:75,a5:100 mM)突变216 h内,血清渗透压无显著变化(P>0.05)；鳃、肠、肾Na+-K+-ATPase和Ca2+-ATPase活力均升高,呈现上升(6 h、72 h和6 h)后下降(24 h、96 h和24 h)并趋于稳定的趋势；血清Na+、Cl和K+含量无显著变化,a3和a4组血清Ca2+含量与对照组无显著差异(P>0.05),a5组血清Ca2+含量72 h后显著增加并维持稳定(P<0.05),a4、a5处理组血清Mg2+含量显著下降并维持稳定(P<0.05),但a3组血清Mg2+含量无显著变化(P>0.05)；光镜结果显示(216 h),各处理组鳃丝氯细胞密度无显著变化,氯细胞大小略有增加,其中a5组显著高于对照组(P<0.05)；电镜结果显示(6 h),a5组氯细胞线粒体内嵴发达,而微管系统出现衰弱现象。Ca2+由自然海水(9.7 mM)向低浓度组(b1：5 mM)突变60 d后,除鳃丝氯细胞密度显著增加(P<0.05)外,其它各项指标均无显著变化。Ca2+由自然海水(9.7 mM)向高浓度组(b3:20, b4:37.5, b5:75 mM)突变60 d后,各处理组血清渗透压均无显著变化；b3和b4组鳃、肠、肾Na+-K+-ATPase和Ca2+-ATPase活力恢复至正常水平,而b5组鳃、肠、肾Na+-K+-ATPase和Ca2+-ATPase活力略有增加,其中鳃和肠Na+-K+-ATPase和Ca2+-ATPase活力显著高于对照组(P<0.05)；各处理组血清Na+、Cl和K-含量无显著变化,血清Ca2+含量与水中Ca2+浓度呈正相关,其中b5组显著高于对照组(P<0.05),而血清Mg2-含量与水中Ca2+浓度呈负相关且均显著低于对照组(P<0.05)；各处理组氯细胞密度均无显著变化(P>0.05),b5组氯细胞大小显著增加(P<0.05),而b3和b4组氯细胞大小无显著变化；b5组氯细胞微管系统密集且管系分支增加。综合分析认为,Ca2+浓度发生突变时,在短期应激反应和长期适应过程中,褐牙鲆幼鱼血清渗透压均无显著变化,但其通过调节鳃、肠和肾Na+-K+-ATPase和Ca2+-ATPase活性及改变血清Ca2+和Mg2+含量来维持体液离子的动态平衡。2、钙镁离子总浓度对褐牙鲆幼鱼血清渗透压的影响及其渗透调节机制的研究钙镁离子由自然海水(60 mM)向低浓度组(A1: 7.5, A2:27 mM)突变216 h内,A1和A2组血清渗透压分别在72 h和6 h显著增加(P<0.05),在其它取样时间无显著变化；鳃Na+-K+-ATPase和Ca2+-ATPase活力呈先下降(6 h)后上升(72 h)再下降(96 h)并趋于稳定的趋势；A1组血清Na+、K-和Cl-含量无显著变化(P>0.05),血清Ca2+含量在48 h内略有下降,72 h后恢复至正常水平,血清Mg2+含量略有增加,在6 h和144 h时显著高于对照组(P<0.05),A2组血清离子含量无显著变化(P>0.05)；光镜结果显示(216 h),A1和A2组氯细胞密度和大小均无显著变化；电镜结果显示(216 h),A1组氯细胞微管系统疏松,线粒体内嵴出现溶解现象。钙镁离子由自然海水(60 mM)向高浓度组(A4:105, A5:180 mM)突变216 h内,A4组血清渗透压在72 h和216 h显著高于对照组(P<0.05),在其它取样时间与对照组无显著差异,A5组血清渗透压在24 h后显著升高并维持稳定(P<0.05)；鳃Na+-K+-ATPase和Ca2+-ATPase活力呈现上升(72h)后下降(96 h)并趋于稳定的趋势；A4组血清离子含量无显著变化(P<0.05),A5组血清Na+、Cl和Ca2+分别在48 h、24 h和6 h后显著增加并趋于稳定(P<0.05),K+在72 h和96 h时显著增加(P<0.05),Mg2+含量下降并维持稳定,在6 h和12 h时显著低于对照组(P<0.05)；光镜结果显示(216 h),A4组氯细胞密度和大小均无显著变化(P>0.05),A5组氯细胞密度无显著变化,而氯细胞大小显著高于其他各组(P<0.05)；电镜结果显示(216 h),A4组氯细胞线粒体体积略有增大,A5组氯细胞线粒体密度显著增加,微管系统管径变粗。钙镁离子由自然海水(60 mM)向低浓度组(B1:27, B2:45 mM)突变60 d后,血清渗透压和血清离子均恢复至正常水平；鳃Na+-K+-ATPase和Ca2+-ATPase活力显著高于对照组(P<0.05)；光镜结果显示,B1组氯细胞密度显著增加(P<0.05),氯细胞大小无显著变化；电镜结果显示,B1组氯细胞线粒体密度显著增加,微管系统发达。钙镁离子由自然海水(60 mM)向高浓度组(B4:90, B5:105 mM)突变60 d后,血清渗透压、血清离子和鳃Na+-K+-ATPase和Ca2+-ATPase活力均无显著变化；光镜结果显示,B5组氯细胞密度显著下降(P<0.05),而氯细胞大小显著增加(P<0.05)；电镜结果显示,B5组氯细胞微管系统分支增加,管径变粗。综合分析认为,钙镁离子浓度发生突变时,在短期应激反应中,低钙镁处理组和高钙镁处理组褐牙鲆幼鱼血清渗透压均显著增加,经60天适应后均恢复至正常水平,褐牙鲆幼鱼主要通过调节其鳃Na+-K+-ATPase和Ca2+-ATPase活性及血清离子含量来维持其体液离子的动态平衡。3、钙镁离子浓度对褐牙鲆幼鱼鳃黏液细胞数量、超微结构及分泌方式的影响褐牙鲆幼鱼在低浓度钙镁离子人工海水中(A1:7.5, A2:27mM)适应9 d后,鳃弓黏液细胞数量增加,但与对照组无显著差异(P>0.05),高浓度钙镁离子组(A4:105, A5:180 mM)黏液细胞数量减少,其中A5组显著低于对照组及各低浓度钙镁离子组(P<0.05)；在低浓度钙镁离子人工海水中(B1：27 mM)适应60 d后,鳃弓黏液细胞数量显著增加(P<0.05),高浓度钙镁离子处理组(B3：105 mM)鳃弓黏液细胞数量减少,但与对照组无显著差异(P>0.05)；在低浓度钙离子人工海水中(C1：1.25 mM)适应9 d后,鳃弓黏液细胞数量增加,各高浓度钙离子组(C3:37.5, C4:75, C5:100 mM)鳃弓黏液细胞数量减少,但各处理组间鳃弓黏液细胞数量无显著差异(P>0.05)；在低浓度钙离子人工海水中(D1：5 mM)适应60 d后,鳃弓黏液细胞数量增加,但与对照组无显著差异(P>0.05),各高浓度钙离子处理组(D3:37.5,D4:75 mM)鳃弓黏液细胞数量减少,其中D4组显著低于D1组(P<0.05),但D3、D4组与对照组均无显著差异(P>0.05)。高浓度钙离子胁迫组鳃弓黏液细胞的数量与对照组无显著差异(P>0.05),且其鳃丝黏液细胞的超微结构相同；高浓度钙离子胁迫组和对照组鳃丝黏液细胞的分泌方式相同,均为顶浆分泌,但胁迫组实验鱼黏液细胞分泌活动旺盛,大量黏液正在向胞外排放,而对照组黏液细胞的黏液分泌量较少。综合分析认为,低浓度钙镁离子或钙离子能够刺激褐牙鲆幼鱼鳃弓黏液细胞的增殖,而高浓度钙镁离子或钙离子则能够抑制黏液细胞的增殖,且褐牙鲆幼鱼对高浓度钙离子胁迫具有较强的耐受能力。4、盐度驯化对褐牙鲆幼鱼血清渗透压和鳃Na+-K+-ATPase活力的影响(1)盐度驯化对血清渗透压的影响：在以不同日盐度变化幅度的低盐和高盐驯化过程中,褐牙鲆幼鱼血清渗透压与驯化到的盐度均呈明显正相关性,随驯化盐度升降而上升或下降。在驯化至高盐47后的适应过程中,以日变幅7驯化的处理组血清渗透压2 d后显著下降并趋于稳定(P<0.05),而以日变幅14驯化的处理组血清渗透压呈逐渐升高趋势；在驯化至低盐5后的适应过程中,以日变幅7驯化的处理组血清渗透压2 d后显著下降并趋于稳定(P<0.05),而以日变幅14驯化的处理组血清渗透压在第2 d时显著下降(P<0.05),4 d后趋于稳定。(2)盐度驯化对鳃丝Na+-K+-ATPase活力的影响：以日变幅3.5驯化至5和47的过程中,Na+-K+-ATPase活力均呈升高趋势,而以日变幅7和14驯化至不同盐度时,Na++K+-ATPase活力均无显著变化(P>0.05)。在驯化至高盐47后的适应过程中,以日变幅7驯化的处理组Na--K+-ATPase活力2 d后显著上升并趋于稳定(P<0.05),而以日变幅14驯化的处理组Na+-K+-ATPase活力呈现上升后下降的趋势,各时间段均无显著差异(P>0.05)；在驯化至低盐5后的适应过程中以日变幅7驯化的处理组Na+-K+-ATPase活力第2 d时显著增加,之后略有下降并趋于稳定,而以日变幅14驯化的处理组Na+-K+-ATPase活力则呈先下降后回升再下降的趋势。综合分析认为,褐牙鲆幼鱼鳃丝Na--K+-ATPase活力在其正常生活的环境中最低,而在高盐驯化和低盐驯化时均升高,此外,以日变幅7的盐度驯化能够缩短褐牙鲆幼鱼在盐度适应过程中的调整时间。更多还原

【Abstract】 Japanese flounder, Paralichthys olivaceus, which is found in the coast of Northeast Asia, such as China, Japan and Korea, is a kind of high-value commercial fish and plays an important role in marine aquaculture. The author studies the effect and the osmoregulation mechanisms of calcium and magnesium concentrations and salinity acclimatization on serum osmolarity of juvenile Japanese flounder. The main results and conclusions from the studies are as follows:1. The effect and the osmoregulation mechanisms of calcium concentration on serum osmolarity of juvenile P. olivaceusThe osmoregulation mechanisms of juvenile Japanese flounder transferred directly from seawater (Ca2+,9.7 mM) to aritificial seawater with different calcium concentrations were studied for 216 hours and 60 days respectively. Changes in serum osmolarity, serum ions, gill, intestine and kidney Na+-K+-ATPase and Ca2+-ATPase activities, as well as the density, size and ultrastructure of chloride cell in gill filament were measured at 6,12,24,72,96,144,216 hour and 60 day. First, changes of experimental parameters were observed in low calcium concentration treatment (a1: 1.25 mM) for 216 h. Serum osmolarity increased significantly at 6 h, and returned to control group level after 12 h. Gill and intestine Na+-K+-ATPase and Ca+-ATPase activities showed transiently decreased at 6 h and 72 h respectively, and quickly returned to control group level at 12 h and 96 h separately. However, kidney Na+-K+-ATPase and Ca+-ATPase activities increased significantly, reached a peak value at 6 h, slightly decreased after 24 h and maintained a new steady state (P<0.05). Serum Na+, Cl- and K+ concentrations showed no significant change (P>0.05). Serum calcium concentration slightly decreased after 6 h and maintained a new steady state, while magnesium concentration increased significantly at 6 h and hold steady (P<0.05). Light microscope results showed that the density and size of chloride cell had no significant change at 216 h (P>0.05), while electron microscope results showed that chloride cell membranous tubules reduced at 6 h. Second, changes of experimental parameters were also observed in high calcium concentration treatments (a3:37.5, a4:75, a5:100 mM) for 216 h. The results showed that serum osmolarity maintained a steady state during the whole experiment (P>0.05). Gill, intestine and kidney Na+-K+-ATPase and Ca2+-ATPase activities increased at 6 h,72 h and 6 h respectively, and then decreased at 24 h,96 h and-24 h separately and hold steady, but higher than control treatment. Serum Na+, Cl- and K+ concentrations showed no significant change (P>0.05). Serum calcium concentration of as increased significantly after 72 h and maintained a new steady state (P<0.05), whlie calcium of a3 and a4 showed no change. Serum magnesium concentration of a4 and as decreased significantly after 6 h and maintained a new steady state (P<0.05), while magnesium of a3 showed no change. Light microscope results showed that the density of chloride cell of the three treatments had no significant change at 216 h (P>0.05), while the size of chloride cell of a5 increased significantly (P<0.05). Electron microscope results showed that the cristae of mitochondria of as were more stronger, but the tubular network became weaker at 6 h. Third, changes of experimental parameters were measured in low calcium concentration treatment (b1:5 mM) at 60 d. The results showed that all of experimental parameters recovered to normal levels except for the density of chloride cell which increased significantly (P<0.05). Last, changes of experimental parameters were also measured in high calcium concentration treatments (b3:20, b4:37.5, b5:75 mM) at 60 d. The results showed that serum osmolarity in the three treatments had no significant change (P>0.05). Gill, intestine and kidney Na+-K+-ATPase and Ca--ATPase activities in b3 and b4 recovered to control group level, while increased in b5. Serum Na+, Cl- and K+ concentrations showed no significant change (P>0.05). Serum calcium concentration was positive with ambient calcium concentration, while serum magnesium concentration was negative with ambient calcium concentration (P<0.05). Light microscope results showed that the density of chloride cell in the three treatments had no significant change (P>0.05), while the size of chloride cell of b5 increased significantly (P<0.05). Electron microscope results showed that chloride cell tubular network of b5 were greater development. We concluded that serum osmolarity of juvenile Japanese flounder had no significant change under short and long adaptive process when calcium concentration abruptly changed, but it could keep ions kinetic equilibrium of body fluid by adjusting Na+-K+-ATPase and Ca2+-ATPase activities in gill, intestine and kidney as well as serum calcium and magnesium concentrations.2. The effect and the osmoregulation mechanisms of calcium and magnesium total concentrations on serum osmolarity of juvenile P. olivaceusThe osmoregulation mechanisms of juvenile Japanese flounder transferred directly from seawater (Ca2++Mg2+:60 mM) to artificial seawater with different calcium and magnesium total concentrations were studied for 216 hours and 60 days respectively. Chages in serum osmolarity, serum ions, gill Na+-K+-ATPase and Ca2+-ATPase activities, as well as the density, size and ultrastructure of chloride cell in gill filament were measured at 6,12,24,48,72,96,144,216 hour and 60 day. First, changes of experimental parameters were measured in low calcium and magnesium total concentrations treatments (A1:7.5, A2:27 mM) for 216 h. Serum osmolarity of A1 and A2 increased significantly only at 72 h and 6 h respectively (P<0.05). Gill Na+-K+-ATPase and Ca2+-ATPase activities decreased at 6 h, but began to increase after 12 h, reached a peak vaule at 72 h, and ruturned to control group level after 96 h. Serum Na+, Cl- and K+ concentrations of A1 had no change (P>0.05), while serum Ca2+ concentration of A1 slightly decreased within 48 h, and returned to control group level after 72 h. Serum Mg2+ concentration of A1 significantly increased at 6 h and 144 h (P<0.05). Serum ions of A2 had no significant change (P>0.05). Light microscope results showed that the density and size of chloride cell of A1 and A2 had no significant change at 216 h (P>0.05). Electron microscope results showed that chloride cell tubular network became loose and the cristae of mitochondria became vague of A1 at 216 h. Second, changes of experimental parameters were also measured in high calcium and magnesium total concentrations treatments (A4:105, A5: 180 mM) for 216 h. Serum osmolarity of A4 significantly increased only at 72 h and 216 h (P<0.05), while serum osmolarity of A5 significantly increased after 24 h and hold steady (P<0.05). Serum ions of A4 had no significant change, while serum Na+ Cl-, and Ca2+ of A5 increased significantly at 48 h,24 h and 6 h respectively and maintained a new steady state (P<0.05). However, serum K+ concentration increased significantly only at 72 h and 96 h (P<0.05). Serum magnesium concentration significantly decreased at 6 h,12 h and maintained at a low state (P<0.05). Light microscope results showed that the density and size of chloride cell of A4 had no significant change, while the size of chloride cell of A5 significantly increased (P<0.05). Electron microscope results showed that the volum of mitochondriale of chloride cell were larger and the tubular network were greater development of A4, and the density of mitochondria were greater abundance of A5. Third, changes of experimental paramters were observed in low calcium and magnesium total concentrations treatments (B1:27, B2:45 mM) at 60 d. Both serum osmolarity and ions returned to normal levels, while gill Na+-K+-ATPase and Ca2+-ATPase activities significantly increased (P<0.05). Light microscope results showed that the density of chloride cell of B1 significantly increased (P<0.05), while the size of chloride cell had no change (P>0.05). Electron microscope results showed that chloride cell mitochondria were more abundance and the tubular network were greater development in B1. Last, changes of experimental parameters were also observed in high calcium and magnesium total concentrations treatments (B4:90, B5:105 mM) at 60 d. Serum osmolarity, serum ions and gill Na+-K+-ATPase and Ca2+-ATPase activities were all no significant change (P>0.05). Light microscope results showed that the density of chloride cell of B5 significantly decreased (P<0.05), while the size of chloride cell significantly increased (P<0.05). Electron microscope results showed that chloride cell mitochondria criatae were stronger, and the tubular network were greater development of B5. We concluded that serum osmolarity of juvenile Japanese flounder increased significantly both in low and high treatments when calcium and magnesium total concentrations changed shortly, but it returned to control group level after long adaptive process by adjusting gill Na+-K+-ATPase and Ca2+-ATPase activities as well as serum ions to keep ions kinetic equilibrium of body fluid.3. The effect of calcium and magnesium concentrations on the number, ultrastructure and secretion of gill mucous cells of juvenile P. olivaceusThis part included 5 experiments. Exp.1, the effect of calcium and magnesium total concentrations on the number of gill arch mucous cells of juvenile Japanese flounder at 9 d. The results showed that the number of mucous cells of A1 (7.5 mM) and A2 (27 mM) increased slightly, but had no significant change comparing with control group (P>0.05). The number of mucous cells of A4(105 mM) and A5 (180 mM) decreased slightly, and it decreased significantly in A5 (P<0.05). Exp.2, the effect of calcium and magnesium total concentrations on the number of gill arch mucous cells of juvenile Japanese flounder at 60 d. The results showed that the number of mucous cells increased significantly in B1 (27 mM) (P<0.05), while decreased in B3 (105 mM). Exp.3, the effect of calcium concentration on the number of gill arch mucous cells of juvenile Japanese flounder at 9 d. The results showed that the number of mucous cells increased in C1 (1.25 mM), and decreased in C3(37.5 mM), C4 (75 mM) and C5 (100 mM), but there was no significant change among different groups (P>0.05). Exp.4, the effect of calcium concentration on the number of gill arch mucous cells of juvenile Japanese flounder at 60 d. The results showed that the number of mucous cells of D1 (5 mM) increased, but had no significant change comparing with control group (P>0.05). The number of mucous cells of D3 (37.5 mM) and D4 (75 mM) decreased, and the D4 were significantly lower than D1 (P<0.05). Exp.5, the effect of high calcium concentration stress on the number, ultrastructure and secretion of mucous cells in the gill of juvenile Japanese flounder. The rusults showed that the number and ultrastructure of mucous cells had no obvious change between stress group and control group. The secretion of mucous cells was same between the two experimental groups, both belonging to apocrine secretion. The mucous cells secretory activity of stress group was vigorous with discharging massive mucus, while it was little in control group. We concluded that low calcium and magnesium total concentraitons or calium concentration could stimulate the proliferation of gill mucous cells, while high calcium and magnesium total concentraitons or calcium concentration’could suppress the proliferation of gill mucous cells. Besides, juvenile Japanese flounder had strong tolerance of high calcium concentration stress.4. The effect of salinity acclimatization on serum osmolarity and gill Na--K--ATPase activity of juvenile P. olivaceusThe osmoregulation mechanisms of juvenile Japanese flounder transferred from seawater (33‰) to high salinity (47‰) and low salinity (5‰) with different (3.5,7, 14) day salinity variations and during adaptive process were studied. Chages in serum osmolarity and gill Na+-K+-ATPase activity were measured every two days in acclimatization and at 1,2,4,8 d in adaptive process. (1) Changes of serum osmolarity were observed. The results showed that serum osmolarity was obviously positive with salinity in acclimatization. Besides, in adaptive process, serum osmolarity significantly decreased in high salinity with 7 day variation and low salinity with 7 and 14 day variations, and reached a steady state at 2 d,2 d and 4 d respevtively. However, serum osmolarity showed increased tendency in high salinity with 14 day variation. (2) Changes of gill Na+-K+-ATPase activity were also observed. The results showed that gill Na+-K+-ATPase activity showed increased tendency both in high and low salinity acclimatization with 3.5 day variation, while it showed no change with 7 and 14 day variation. In adapted process, gill Na+-K+-ATPase activity significantly increased at 2 d and maintained a new steady state in high salinity with 7 day variation, and the same phenomenon was observed in low salinity with 7 day variation. Gill Na+-K+-ATPase activity showed increase-decrease tendency in high salinity with 14 day variation, but there was no significant change. Gill Na+-K+-ATPase activity showed decrease-increase-decrease tendency in low salinity with 14 day variation. We concluded that gill Na+-K+-ATPase activity of juvenile Japanese flounder was lowest in seawater which it natural lived while increased both in high and low salinity acclimatization. Besides, the adaptive time of juvenile Japanese flounder was reduced during adapted process by 7 day variaiton.更多还原