低盐度海水和淡水对中华绒螯蟹性腺发育及交配行为的影响

研究了低盐度海水(盐度为15)和淡水(盐度为0)对中华绒螯蟹(Eriocheir sinensis,以下简称河蟹)性腺发育及交配行为的影响,并比较了河蟹交配和产卵前后的性腺指数及肝胰腺指数的变化。实验分为4组,分别为低盐度海水雌蟹组、淡水雌蟹组、低盐度海水雄蟹组和淡水雄蟹组。结果表明,(1)各组河蟹的成活率均在80%左右,无显著差异(P>0.05);(2)实验第15天、30天和45天时,低盐度海水雌蟹组的卵巢指数显著高于淡水雌蟹组(P<0.05),低盐度海水雄蟹组的性腺指数也略高于淡水雄蟹组,但差异不显著(P>0.05);(3)实验第30天时,低盐度海水雌蟹组的肝胰腺指数显著低于淡水雌蟹组(P<0.05),其余采样时间两组雌体间或两组雄体间的肝胰腺指数差异不显著(P>0.05),实验期间,两组雌体的肝胰腺指数均显著下降(P<0.05);(4)实验第45天,低盐度海水雌蟹组和雄蟹组实验个体全部能够交配,有66.7%的低盐度海水雌蟹组的个体交配后2 d内产卵,淡水雄蟹组有部分个体在低盐度海水中有发情行为;(5)低盐度海水组,雌蟹产卵后和雄蟹交配后的性腺指数均显著下降(P<0.05),但肝胰腺指数下降不显著(P>0.05)。     

盐度对日本鳗鲡(Anguilla japonica)渗透压调节的影响

以日本鳗鲡(505.1±35.7)g为实验对象,分别在淡水(盐度0)、盐度10和盐度33条件下处理14 d,在0、1、4、12、24、96 h和14 d时测定其血清渗透压、离子(Na+、K+、Cl-)浓度和鳃Na+/K+-ATP酶活力指标。结果表明:日本鳗鲡血清等渗点为329.1 m Osm·kg-1,其对应盐度为10.48;随着处理时间延长,盐度处理组血清渗透压、Na+和Cl-浓度均呈现先上升后下降的趋势;血清K+浓度受盐度影响较小(P0.05);盐度10处理组鳃Na+/K+-ATP酶活力于12 h达到最小值(5.40±0.72)μmol·mg-1·h-1,至96 h时恢复至淡水组水平(P0.05);而盐度33处理组鳃Na+/K+-ATP酶活力则表现为先快速下降,后快速上升,并于24 h达到最大值(13.05±0.62)μmol·mg-1·h-1,约为淡水组的1.5倍(P0.05)。日本鳗鲡的渗透压调节可初步分为3个阶段:(1)快速升高期,血清渗透压、Na+和Cl-浓度异常升高,鳃Na+/K+-ATP酶活力受到抑制;(2)缓慢升高期,鱼体补偿失水以缓解渗透压升高,血清渗透压、Na+和Cl-浓度表现为缓慢升高,鳃Na+/K+-ATP酶被激活;(3)适应期,鳃Na+/K+-ATP酶活力处于较高水平,血清渗透压、离子浓度基本恢复。     

盐度胁迫对红耳龟生长与血液生化指标的影响

为了解外来物种红耳龟在不同盐度水域中的生存状况,本研究选用体重67.28 g±19.39 g的红耳龟进行为期70 d的不同盐度胁迫实验,分别测定红耳龟在对照组以及盐度为10‰组和20‰组(以下简写为10组和20组)的体重特定增长率和血液生化指标变化.结果表明,盐度10组的体重特定增长率极显著高于对照组和盐度20组(P＜0.01),而盐度20组的体重特定增长率略大于对照组,但差异不显著(P＞0.05);盐度10组的肌酸激酶(CK)、谷草转氨酶(AST)、乳酸脱氢酶(LDH)、碱性磷酸酶(ALP)的活性显著高于对照组(P＜0.05);盐度10组和20组的血糖(Glu)含量显著高于对照组(P＜o.05);各盐度组血清渗透压(Osmp)、Na+、C1、K+、Mg2+、血清尿素氮(BUN)、尿酸(UA)含量差异显著(P＜0.05);盐度20组Ca2+显著高于对照组(P＜0.05).说明红耳龟可通过提高血液中血糖含量及代谢所需的酶活性使得其代谢水平升高,从而为抵抗胁迫提供所需能量;还可以通过提高血液渗透压及无机离子的浓度来适应外界渗透压的升高,从而使其能够在不同盐度水域中生存.本研究为红耳龟对盐度的耐受生理及入侵机理研究提供生理学方面的依据.     

混合植物油替代鱼油对中华绒螯蟹成体雄蟹常规成分和脂肪酸组成的影响

为研究育肥饲料中混合植物油替代鱼油对中华绒螯蟹(Eriocheir sinensis)成体雄蟹常规成分和脂肪酸组成的影响,采用豆油和菜籽油混合物替代鱼油制成5种不同鱼油替代水平(0%、25%、50%、75%和100%)的等氮等脂育肥饲料(分别记为1#~5#饲料组)饲喂雄蟹,测定5组雄蟹肝胰腺、肌肉和性腺中的常规成分和脂肪酸组成,并对实验数据进行方差分析。结果显示:(1)饲料1#组性腺灰分含量显著高于饲料4#和5#组(P0.05),但各组性腺中的水分、总脂和蛋白含量均无显著差异(P0.05);饲料1#组肝胰腺中的水分和灰分最高,但其总脂含量低于其他组,各组肝胰腺的蛋白含量无显著差异(P0.05);除1#饲料组外,肌肉中的总脂和灰分含量随鱼油替代水平的升高而显著上升(P0.05),而水分和蛋白含量均无显著差异(P0.05)。(2)各饲料组精巢中总饱和脂肪酸(∑SFA)、总多不饱和脂肪酸(∑PUFA)和总高度不饱和脂肪酸(∑HUFA)含量无显著差异(P0.05),其总n-6多不饱和脂肪酸(∑n-6PUFA)含量随鱼油替代水平升高而升高,而总n-3多不饱和脂肪酸(∑n-3PUFA)含量和∑n-3PUFA/∑n-6PUFA比值呈显著下降趋势(P0.05)。(3)肝胰腺中∑n-3PUFA和∑HUFA含量具有显著的组间差异,且均以饲料3#组最高。但各组的∑PUFA和∑n-6PUFA含量差异并不显著(P0.05)。(4)肌肉中大部分脂肪酸组成无显著差异,仅∑n-6PUFA含量随鱼油替代水平升高而升高。综上,中华绒螯蟹育肥饲料中植物油(豆油与菜籽油含量为1︰1)替代鱼油对成体雄蟹可食组织中水分和蛋白含量并无显著影响,但会对其脂肪酸组成造成显著的影响,50%的鱼油替代水平有利于雄蟹肝胰腺和肌肉中的脂肪沉积。     

不同盐度条件下中华绒螯蟹亲蟹行为及血淋巴生理变化

设定淡水对照组、盐度18适应组、盐度30骤变组(18→30)和盐度0骤变组(18→0),采用视频记录分析法研究了不同盐度条件下中华绒螯蟹雌性亲蟹的8项行为学指标变化,并测定了血淋巴渗透压及离子、血蓝蛋白含量。结果表明:中华绒螯蟹亲蟹封闭反应行为仅发生于盐度组(盐度18组和盐度30骤变组),且盐度30骤变组封闭反应时间显著高于盐度18组(P<0.05);腹部开合行为仅见于盐度0骤变组;盐度组第一触角回缩时间显著高于对照组(P<0.05);其他5项行为学指标活动频率均于盐度0骤变组最高。各实验组亲蟹血淋巴渗透压及离子浓度均高于外界实验水体,且均随盐度升高而增大。血蓝蛋白含量随盐度的降低而升高,盐度0骤变组血蓝蛋白含量显著高于盐度30骤变组(P<0.05)。分析认为,中华绒螯蟹亲蟹在0～30盐度范围内进行高渗透压调节,腹部开合行为是其在低盐度环境下暴露尾肠吸收水中离子的一种行为策略,封闭反应有助于机体减少高盐度坏境下水的吸收及扩散失盐。     

急性高渗胁迫对中华绒螯蟹雄蟹组织中可溶性蛋白质、血蓝蛋白、血糖与肝糖原含量的影响

实验以淡水(0.3‰)处理组为对照,研究了急性高渗胁迫(盐度16‰和30‰)对中华绒螯蟹(Eriocheirsinensis)雄蟹血淋巴和肝胰腺中可溶性蛋白质、血蓝蛋白、血糖及肝糖原含量的影响。结果显示,与对照组相比,进入16‰及30‰盐度后,蟹的血淋巴中可溶性蛋白质含量从6—48h显著降低(P<0.05),至72h时显著增加(P<0.05),而机体肝胰腺的可溶性蛋白质含量从12h开始至96h显著降低(P<0.05);血蓝蛋白含量在前24h显著降低(P<0.05),48h开始显著升高(P<0.05);肝糖原含量在6—96h与对照组相比有显著的降低(P<0.05);16‰盐度组蟹的血糖含量在24—48h时显著下降(P<0.05),到72h开始逐渐恢复(P>0.05),而30‰盐度组其血糖含量早在6—12h时就有显著的下降(P<0.05),到24h则开始逐渐升高。研究表明,在急性盐度胁迫下,糖类和蛋白质对中华绒螯蟹渗透压调节起重要的作用,其优先利用糖类的分解获得所需的能量,其中盐度越高,机体内葡萄糖的消耗越快,血糖水平的恢复也相应较快。对蛋白质的利用稍次之,机体可利用蛋白质的分解获得其渗透压调节的效应物游离氨基酸以维持渗透压平衡。     

饲料中添加合成虾青素对中华绒螯蟹成体雌蟹性腺发育、色泽和抗氧化能力的影响

采用在基础饲料中分别添加0、130和260 mg/kg的合成虾青素配制成3种粗蛋白和粗脂肪含量分别为42%和16%的等氮等脂的中华绒螯蟹(Eriocheir sinensis)育肥饲料(分别记为饲料1、2和3), 以中华绒螯蟹商业育肥饲料作为饲料4, 分别投喂4组雌蟹(每个饲料组3个重复水槽, 每个水槽中12只蟹), 进行为期60d的室内养殖实验, 以探讨添加合成虾青素对中华绒螯蟹成体雌蟹性腺发育、色泽及抗氧化能力的影响。结果显示: (1)实验进行到30d和60d, 在饲料中添加虾青素对雌体肝胰腺指数(HSI)和性腺指数(GSI)均无显著影响(P>0.05)。(2)性腺、肝胰腺和头胸甲中的总类胡萝卜素含量和红度值(a值)均以饲料3组最高, 性腺亮度值(L值)和黄度值(b值)以饲料1组最高(P<0.05); 饲料2组头胸甲的b值最高, 饲料1组最低(P<0.05)。(3) 饲料1组中华绒螯蟹血清过氧化物酶(POD)活性显著高于其他组, 饲料4组的过氧化氢酶(CAT)、谷胱甘肽还原酶(GR)、乳酸脱氢酶(LDH)、总抗氧化能力(T-AOC)、丙二醛(MDA)和乳酸(LD)含量最高(P<0.05); 肝胰腺中的SOD、T-AOC、GSH-Px和GR活性均以饲料1组最高, 饲料2组最低(P<0.05)。(4) 饲料2组血清酸性磷酸酶(ACP)活性和血蓝蛋白(Hc)含量显著高于其他组, 其余各组间差异不显著。血清中的碱性磷酸酶(ALP)和γ-谷氨酰转移酶(γ-GT)活性和一氧化氮(NO)含量均以饲料4组最高(P<0.05), 肝胰腺中的ACP、ALP和γ-GT活性以饲料1组最高。综上, 在育肥饲料中添加合成虾青素对成体雌蟹性腺发育无显著影响, 但可显著提高头胸甲、肝胰腺和卵巢中的类胡萝卜素总量、色泽和抗氧化能力, 建议雌蟹育肥饲料中合成虾青素的含量为90 mg/kg左右。     

饲料中添加雨生红球藻粉对中华绒螯蟹成体雄蟹生化组成的影响

为研究饲料中添加雨生红球藻(Haematococcus pluvialis)粉对中华绒螯蟹(Eriocheir sinensis)成体雄蟹成活、增重和生化组成的影响,分别在饲料中添加0、0.2%、0.4%和0.6%的雨生红球藻粉,配制4种等氮等脂的育肥饲料,投喂生殖蜕壳后雄蟹60 d,计算各组成活率、增重率、特定生长率和肥满度,同时测定了组织中的常规营养成分、脂肪酸组成和类胡萝卜素含量,并对实验数据进行方差分析。结果显示:(1)饲料中添加雨生红球藻粉对成体雄蟹的成活率、增重率、特定生长率和肥满度均无显著影响。(2)雄蟹性腺中的粗蛋白及肝胰腺总脂含量均以雨生红球藻粉0.4%组最高,而肌肉水分含量随饲料中雨生红球藻粉含量的升高整体呈上升趋势(P0.05)。(3)性腺中的脂肪酸C20:0含量随饲料雨生红球藻粉添加水平的升高而显著上升,而C16:1n7、C18:2n6和C18:3n3含量分别在雨生红球藻粉0.6%组、0.4%组和0组最高(P0.05)。(4)肝胰腺中的脂肪酸C14:0、C18:0和C18:1n7含量均以雨生红球藻粉0.2%组最高,而C18:1n9和总单不饱和脂肪酸(∑MUFA)含量均以雨生红球藻粉0.6%组最高(P0.05)。(5)肌肉中的脂肪酸C14:1n5和C20:2n6含量在雨生红球藻粉0.2%组最高(P0.05),C16:0含量及二十二碳六烯酸/二十碳五烯酸(DHA/EPA)比例以雨生红球藻粉0.4%组最高,而C22:6n3、总多不饱和脂肪酸(∑PUFA)、∑n-3 PUFA和总高度不饱和脂肪酸(∑HUFA)含量均以雨生红球藻粉0.6%组最高(P0.05)。(6)肝胰腺中的虾青素、β-胡萝卜素和头胸甲中的虾青素、叶黄素、玉米黄素含量随饲料中雨生红球藻粉含量的升高而显著上升(P0.05)。综上,饲料中添加雨生红球藻粉对成体雄蟹成活率、增重率和肥满度无显著影响,但可提高性腺粗蛋白、肌肉总多不饱和脂肪酸、肝胰腺和头胸甲中的类胡萝卜素含量,雄蟹育肥饲料中适宜的雨生红球藻粉添加量建议为0.4%左右。     

三种饵料模式对中华绒螯蟹蟹种早期养殖性能、非特异免疫性能及抗病力的影响

为评估不同投喂模式对中华绒螯蟹(Eriocheir sinensis)蟹种质量的影响,采用养殖实验、非特异性免疫指标分析和攻毒实验方法,比较了配合饲料、传统饵料和混合投喂模式培育扣蟹在成蟹养殖早期的养殖性能、免疫性能和攻毒后死亡率的变化。所检测的非特异性免疫指标包括碱性磷酸酶(ALP)、酸性磷酸酶(ACP)、γ-谷氨酰转移酶(γ-GT)、总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)、酚氧化酶(PO)、过氧化物酶(POD)、丙二醛(MDA)、一氧化氮(NO)、谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽还原酶(GR)及血蓝蛋白(Hc)活性或含量。采用毒力较强的嗜水气单胞菌(Aeromonas hydrophilia)Y-2-L-1菌株进行攻毒实验。结果显示:(1)无论雄蟹还是雌蟹,配合饲料组第一次蜕壳间期均短于传统投喂和混合投喂模式,且配合饲料组增重率(WGR)及体重特定增长率(SGR)均显著高于另外两组(P0.05);(2)就肝胰腺中非特异性免疫指标而言,无论雄蟹还是雌蟹,配合饲料组γ-谷氨酰转移酶(γ-GT)、总抗氧化能力(T-AOC)和超氧化物歧化酶(SOD)活性均高于另外两组,其中γ-谷氨酰转移酶(γ-GT)差异显著;传统投喂组酸性磷酸酶(ACP)、丙二醛(MDA)、一氧化氮(NO)和过氧化物酶(POD)含量均高于另外两组,其中酸性磷酸酶(ACP)和一氧化氮(NO)含量差异显著;(3)就血清中各免疫指标而言,配合饲料组谷胱甘肽过氧化物酶(GSH-Px)和总抗氧化能力(T-AOC)活性高于另外两组,但差异均不显著(P0.05),传统投喂组碱性磷酸酶(ALP)、酸性磷酸酶(ACP)、丙二醛(MDA)及血蓝蛋白(Hc)等指标含量均高于另外两组,其中丙二醛(MDA)差异显著;(4)无论雄蟹还是雌蟹,传统投喂组扣蟹攻毒后12~96 h的累计死亡率一直高于配合饲料组和混合投喂组,配合饲料组和混合投喂组雄体攻毒后的累计死亡率接近,混合投喂组雌体累计死亡率居于传统投喂组和配合饲料组之间。综上,投喂配合饲料可以提高扣蟹在成蟹早期的养殖性能、免疫性能和攻毒后的成活率,有利于提高蟹种质量。     

育肥饲料中植物油混合替代鱼油对中华绒螯蟹雄体脂肪酸代谢相关基因表达的影响

为研究植物油替代鱼油对中华绒螯蟹(以下简称河蟹)脂肪酸代谢产生的影响, 通过荧光定量PCR (qRTPCR)技术研究了河蟹脂肪酸碳链延长酶(Es-FAE)、9脂肪酸去饱和酶(Es-FAD9)、6脂肪酸去饱和酶b (Es-FAD6b)、脂肪酸结合蛋白3 (Es-FABP3)、脂肪酸结合蛋白9 (Es-FABP9)和脂肪酸结合蛋白10 (Es-FABP10)基因在不同组织中的表达情况, 以及育肥饲料中不同鱼油替代水平(鱼油替代水平分别为0、25%、50%、75%和100%, 记为饲料1#5#组)对河蟹雄体肝胰腺中这些基因mRNA表达水平的影响, 以探讨育肥中鱼油替代对河蟹雄体成蟹脂肪酸代谢的影响。结果表明: (1) Es-FAE、Es-FAD6b和Es-FAD9均在肝胰腺中表达水平最高, 在心脏和血淋巴中表达水平较低, 不同基因在其他组织中的表达规律有所不同; Es-FABP3、Es-FABP9和Es-FABP10主要在肝胰腺、肌肉、输精管和副性腺中表达水平较高, 在胃、肠和心脏中表达水平较低。(2) 随着饲料中鱼油替代水平的提高, 肝胰腺中Es-FAE-mRNA有显著增加趋势; 就Es-FAD6b而言, 饲料3#组显著高于饲料2#组, 其余各组间差异不显著; 随着饲料中鱼油水平的降低, 饲料1#3#组肝胰腺中Es-FAD9-mRNA也有显著增加趋势, 但差异不显著。(3) 就Es-FABP3而言, 饲料2#和3#组肝胰腺中表达水平最高,且显著高于饲料1#组(P0.05), 其余各组间差异不显著; 随着鱼油替代水平的升高, 肝胰腺中Es-FABP9和Es-FABP10-mRNA水平均有先上升后下降趋势, 但各组间差异不显著。综上, 河蟹脂肪酸代谢和转运相关基因在肝胰腺中表达水平最高, 饲料中不同鱼油替代水平对肝胰腺中上述基因mRNA表达水平具有较大影响。     

急性盐度胁迫对军曹鱼稚鱼渗透压调节的影响

研究了环境盐度急性胁迫对军曹鱼(Rachycentron canadum)稚鱼鳃Na⁺-K⁺ATPase(NKA)活性及血清渗透压、Na⁺、K⁺和Cl^-离子调节的影响.结果表明:将稚鱼从盐度37中直接转移至盐度0、5、15、25、37（对照）和45的水体中,12 h后仅盐度0处理出现死亡(死亡率100％).各处理鳃NKA活性和血清渗透压在最初3 h内出现一定波动,随后变化平稳.试验结束时(12 h), NKA活性与盐度梯度呈“U”型分布,盐度5处理酶活性显著高于其它处理(P<0.05),盐度15处理活性最低,而各处理的血清渗透压大小(293～399 mOsmol·kg^-1)与盐度呈正相关;在3～12 h内稚鱼血清Na⁺和Cl^-浓度随盐度升高而升高,但增幅较小,血清K⁺浓度则与盐度呈负相关;12 h稚鱼的等渗点为328.2 mOsm·kg^-1,相当于盐度11.48,而Na⁺、K⁺和Cl^-等离子点分别为155.2、6.16和137.1 mmol·L^-1,分别相当于盐度10.68、20.44及8.41.军曹鱼在生理上具有广盐性鱼类的“低渗环境高NKA活性”特征,有较强及迅速的渗透压和离子调节与平衡能力.     

Branchial osmoregulatory response to salinity in the gilthead sea bream, Sparus auratus

The branchial osmoregulatory response of gilthead sea bream (Sparus auratus L.) to short-term (2-192 hr) and long-term (2 weeks) exposure to different environmental salinities (5 per thousand, 15 per thousand, 25 per thousand, 38 per thousand and 60 per thousand) was investigated. A "U-shaped" relationship was observed between environmental salinity and gill Na+,K+ -ATPase activity in both long- and short-term exposure to altered salinity, with the increase in activity occurring between 24 and 96 hr after the onset of exposure. Plasma osmolality and plasma ions (sodium, chloride, calcium and potassium) showed a tendency to increase in parallel with salinity. These variables only differed significantly (P<0.05) in fish adapted to 60 per thousand salinity with respect to fish adapted to full-strength sea-water (SW). Plasma glucose remained unchanged whereas plasma lactate was elevated at 5 per thousand and 60 per thousand. Muscle water content (MWC) was significantly lower in fish adapted to 60 per thousand. Chloride cells (CC) were only present on the surface of the gill filaments and absent from the secondary lamellae. CC distribution was not altered by external salinity. However, the number and size of CC were significantly increased at salinity extremes (5 per thousand and 60 per thousand), whereas fish exposed to intermediate salinities (15 per thousand and 25 per thousand) had fewer and smaller cells. Furthermore, the CC of fish exposed to diluted SW became rounder whereas they were more elongated in fish in full-strength and hypersaline SW. This is consistent with previous reports indicating the existence of two CC types in euryhaline fish. At likely environmental salinities, gilthead sea bream show minor changes in plasma variables and the effective regulation of gill Na+,K+ -ATPase. However, at very low salinities both haemodilution and up-regulation of gill Na+,K+ -ATPase predict a poor adaptation most likely related to deficiency or absence of specific components of the CC important for ion xuptake.     

Expression profiles of Na+,K+-ATPase during acute and chronic hypo-osmotic stress in the blue crab Callinectes sapidus

During acclimation to dilute seawater, the specific activity of Na+,K+-ATPase increases substantially in the posterior gills of the blue crab Callinectes sapidus. To determine whether this increase occurs through regulation of pre-existing enzyme or synthesis of new enzyme, mRNA and protein levels were measured over short (<24 h) and long (18 days) time courses. Na+,K+-ATPase expression, both mRNA and protein, did not change during the initial 24-h exposure to dilute seawater (10 ppt salinity). Thus, osmoregulation in C. sapidus during acute exposure to low salinity likely involves either modulation of existing enzyme or mechanisms other than an increase in the amount of Na+,K+-ATPase enzyme. However, crabs exposed to dilute seawater over 18 days showed a 300% increase in Na+,K+-ATPase specific activity as well as a 200% increase in Na+,K+-ATPase protein levels. Thus, it appears that the increase in Na+,K+-ATPase activity during chronic exposure results from the synthesis of new enzyme. The relative amounts of mRNA for the alpha-subunit increased substantially (by 150%) during the acclimation process, but once the crabs had fully acclimated to low salinity, the mRNA levels had decreased and were not different from levels in crabs fully acclimated to high salinity. Thus, there is transient induction of the Na+,K+-ATPase mRNA levels during acclimation to dilute seawater.     

Ontogeny of salinity tolerance and hyper-osmoregulation by embryos of the intertidal crabs Hemigrapsus edwardsii and Hemigrapsus crenulatus (Decapoda, Grapsidae): survival of acute hyposaline exposure

The adults of Hemigrapsus edwardsii and Hemigrapsus crenulatus are euryhaline crabs and strong hyper-osmoregulators. Their embryos are carried externally attached to the abdominal pleopods of female crabs, where they are exposed to temporal and spatial changes in salinity associated with their intertidal and estuarine habitats. Although embryos lack the branchial and excretory organs responsible for adult osmoregulation, post-gastrula embryos were highly tolerant of exposure to hypo-osmotic sea water. Detached eggs (embryos+envelopes), of both species, at all developmental stages between gastrulation and hatching, exhibited 80-100% survival for periods up to 96 h in sea water (osmolality, 1050 mmol kg(-1)) and in dilutions to 50%, 10%, and 1%. Cleavage stages were less tolerant of dilution; H. edwardsii, <50% survived 24 h in 10% sea water; H. crenulatus <50% survived 6 h in 10% sea water. Post-gastrulation stages strongly hyper-osmoregulated but cleavage stages were hyper-osmoconformers (maintaining internal osmolality approximately 150 mmol kg(-1) above external). Osmoregulatory capacity was reduced just prior hatching, particularly in H. crenulatus, although salinity tolerance remained high. Gastrulation therefore marks a critical stage in the ontogeny of osmoregulation and salinity tolerance. Total Na+/K(+)-ATPase activity increased greatly during embryogenesis of H. crenulatus (undetectable in blastulae; gastrulae 0.31+/-0.05 pmol P(i) embryo(-1) min(-1); pre-hatching 16.4+/-1.0 pmol P(i) embryo(-1) min(-1)). Na+/K(+)-ATPase activity increased in embryos exposed to dilute sea water for 24 h implicating regulation of this transporter in a short-term acclimation response.     

Physiological response in the European flounder (Platichthys flesus) to variable salinity and oxygen conditions

Physiological mechanisms involved in acclimation to variable salinity and oxygen levels and their interaction were studied in European flounder. The fish were acclimated for 2 weeks to freshwater (1 per thousand salinity), brackish water (11 per thousand) or full strength seawater (35 per thousand) under normoxic conditions (water Po(2) = 158 mmHg) and then subjected to 48 h of continued normoxia or hypoxia at a level (Po(2) = 54 mmHg) close to but above the critical Po(2). Plasma osmolality, [Na(+)] and [Cl(-)] increased with increasing salinity, but the rises were limited, reflecting an effective extracellular osmoregulation. Muscle water content was the same at all three salinities, indicating complete cell volume regulation. Gill Na(+)/K(+)-ATPase activity did not change with salinity, but hypoxia caused a 25% decrease in branchial Na(+)/K(+)-ATPase activity at all three salinities. Furthermore, hypoxia induced a significant decrease in mRNA levels of the Na(+)/K(+)-ATPase alpha1-subunit, signifying a reduced expression of the transporter gene. The reduced ATPase activity did not influence extracellular ionic concentrations. Blood [Hb] was stable with salinity, and it was not increased by hypoxia. Instead, hypoxia decreased the erythrocytic nucleoside triphosphate content, a common mechanism for increasing blood O(2) affinity. It is concluded that moderate hypoxia induced an energy saving decrease in branchial Na(+)/K(+)-ATPase activity, which did not compromise extracellular osmoregulation.     

Effects of long-term exposure to different salinities on the location and activity of Na+-K+-ATPase in the gills of juvenile mitten crab, Eriocheir sinensis

The euryhalinity of mitten crab, Eriocheir sinensis, is based on osmoregulation, and thus on the activity of Na(+)-K(+)-ATPase. We studied location and activity of this enzyme in gills of juvenile crabs exposed to 5 per thousand, 25 per thousand, and 40 per thousand salinity. The posterior gills showed always a high number of immunopositive cells (IPC), staining with fluorescent antibody against Na(+)-K(+)-ATPase, covering at 5 per thousand the entire lamellae. At 25 per thousand, they showed fewer IPC which occurred only at the bases of the lamellae. Enzyme activity was consistently higher in posterior than in anterior gills. Low salinity stimulated the activity only in posterior gills. Both histochemical and enzymatic results are consistent with previous ultrastructural observations showing that the epithelial cells of the posterior, but not the anterior gills exhibit typical traits of ionocytes. While an increase in Na(+)-K(+)-ATPase activity at a reduced salinity is consistent with a strong hyper-osmoregulatory capacity in juvenile crabs, a low activity at an enhanced salinity suggests a physiological response, directed towards a reduction of Na(+) uptake. The activity increase of ion-transporting enzymes is directly related to spatial changes in their distribution along the osmoregulatory tissue, i.e. an enhanced number of IPC scattered along the entire lamellae. In juveniles, this allows for successful development and growth at reduced salinities.     

Rapid modulation of gill Na+ + K+-dependent ATPase activity during acclimation of the killifish Fundulus heteroclitus to salinity change.

The enzymatic properties of membrane-bound Na+ + K+-ATPase from gills of killifish acclimated to fresh water, to 16% sea water, or to 30% sea water appear to be identical, indicating that the same enzyme may function to absorb Na+ in low salinities and excrete Na+ at the gills in high salinities. Ammonium ion is an effective substitute for K+: in the ATPase reaction itself, in blocking phosphorylation of the ATPase protein, and in inhibiting the binding of ouabain to the enzyme. The specific activities of the Na+ + K+-ATPase in the three different salinities are consistent with the expected Na+ pumping rates: higher in fresh water and 30% sea water than in 16% sea water. Within one-half hour after transfer of killifish from one salinity to another, gill Na+ + K+-ATPase activities reach equilibrium levels. The rapid increase in Na+ + K+-ATPase activity in gill microsomes of fish acclimating from fresh water to 30% sea water is accompanied by a slow decrease in the number of binding sites for ouabain, supporting the idea that acclimation to short-term salinity changes may involve modifications in the catalytic rate rather than the number of Na+ + K+-ATPase molecules.     

Osmoregulatory action of 17beta-estradiol in the gilthead sea bream Sparus auratus

The osmoregulatory action of 17beta-estradiol (E2) was examined in the euryhaline teleost Sparus auratas. In a first set of experiments, fish were injected once with vegetable oil containing E2 (1, 2 and 5 microg/g body weight), transferred 12h after injection from sea water (SW, 38 ppt salinity) to hypersaline water (HSW, 55 ppt) or to brackish water (BW, 5 ppt salinity) and sampled 12h later (i.e. 24 h post-injection). In a second experiment, fish were injected intraperitoneally with coconut oil alone or containing E2 (10 microg/g body weight) and sampled after 5 days. In the same experiment, after 5 days of treatment, fish of each group were transferred to HSW, BW and SW and sampled 4 days later (9 days post-implant). Gill Na+,K+ -ATPase activity, plasma E2 levels, plasma osmolality, and plasma levels of ions (sodium and calcium), glucose, lactate, protein, triglyceride, and hepatosomatic index were examined. Transfer from SW to HSW produced no significant effects on any parameters assessed. E2 treatment did not affect any parameter. Transfer from SW to BW resulted in a significant decrease in plasma osmolality and plasma sodium but did not affect gill Na+,K+ -ATPase activity. A single dose of E2 attenuated the decrease in these parameters after transfer from SW to BW, but was without effect on gill Na+,K+ -ATPase activity. An implant of E2 (10 microg/g body weight) for 5 days significantly increased plasma calcium, hepatosomatic index, plasma metabolic parameters, and gill Na+,K+ -ATPase activity. In coconut oil-implanted (sham) fish, transfer from SW to HSW or BW during 4 days significantly elevated gill Na+,K+ -ATPase. Gill Na+,K+ -ATPase activity remained unaltered after transfer of E2-treated fish to HSW or BW. However, in E2-treated fish transferred from SW to SW (9 days in SW after E2-implant), gill Na+,K+ -ATPase activity decreased with respect to HSW- or BW-transferred fish. Shams transferred to HSW showed increased levels of lactate, protein, and trygliceride in plasma, while those transferred to BW only displayed increased trygliceride levels. E2-treated fish transferred to HSW showed higher protein levels without any change in other plasmatic parameters, while those transferred to BW displayed elevated plasma glucose levels but decreased osmolality and protein levels. These results substantiate a chronic stimulatory action of E2 on gill Na+,K+ -ATPase activity in the euryhaline teleost Sparus auratas.