【作者】 王兰梅；

【导师】 赵云龙；

【作者基本信息】 华东师范大学 ， 动物学， 2013， 博士

【摘要】 红螯光壳螯虾(Cherax quadricarinatus)又称澳洲淡水龙虾或红螯螯虾,原产于澳大利亚,具有食性杂、生长快、适应性强、个体大、可纯淡水养殖及市场价格高等优点。我国自1992年引入以来,先后在多地试养,也开展了一些科学研究,但至今未形成大规模养殖。其关键在于苗种的供应问题,涉及亲虾精子和卵子成熟的同步率差以及产卵量、孵化率和幼虾成活率低等原因。而目前对其研究主要集中在幼虾的营养需求上,对亲虾繁殖阶段的研究十分有限。而甲壳动物亲体的营养直接影响到卵子的质量,胚胎发育以及幼虾的存活率。因此,本研究从亲体培育环节入手,综合营养学、生理学和分子生物学的理论和方法,辅以电镜技术,较为系统的研究了磷脂营养对雌性红螯光壳螯虾卵黄发生的影响,并从细胞水平和分子水平对其营养调控卵黄发生的生理生化机制和分子机制进行了初步探讨。以期为深入研究虾蟹生殖生理学和规模化红螯光壳螯虾早繁苗种生产等提供基础资料。主要研究结果和结论如下：1.饲料中大豆磷脂对红螯光壳螯虾卵黄发生期生长及卵巢和肝胰腺生化组成的影响为研究饲料中不同水平的大豆粉末磷脂(SL)对红螯光壳螯虾卵黄发生的影响,试验设计了5种实用饲料分别包含0%(Diet1),1%(Diet2),2%(Diet3),4%(Diet4)和6%(Diet5)的SL,饲喂红螯光壳螯虾8周,观察对其卵黄发生期生长以及肝胰腺和卵巢生化组成的影响。虾初始体重25.64±1.53g,每个养殖缸放养8只,每组4个重复。结果表明：饲料中不同水平的SL对卵黄发生期红螯光壳螯虾的成活率和体增重(Weight gain WG)无显著影响(P>0.05),饲喂≥2%SL饲料的虾具有显著高的性腺指数(Gonadosomatic index GSI)(P<0.05),且GSI和肝胰腺指数(Hepatosomatic index HSI)反相关,虾的HSI随饲料中SL的增加呈下降的趋势(P>0.05)。虾肝胰腺中的脂肪酸可能主要来源于饲料,卵巢中多不饱和脂肪酸(Polyunsaturated fatty acid PUFA)和高不饱和脂肪酸(Highlyunsaturated fatty acid HUFA)的含量随饲料中SL的增加而显著升高(P<0.05)。此外,饲料中的亚油酸和亚麻酸相对于其他脂肪酸对红螯光壳螯虾的卵黄发生更为重要,且此阶段可能存在脂类物质从肝胰腺向卵巢的转运。以上结果表明：饲料中的SL对红螯光壳螯虾的卵黄发生具有促进作用。2.饲料中大豆磷脂对红螯光壳螯虾卵黄发生期肝胰腺和卵巢超微结构及脂肪酸结合蛋白(FABP)和卵黄蛋白原(Vg) mRNA表达的影响那么饲料中的SL对红螯光壳螯虾卵黄发生的促进作用是通过什么途径完成的呢?是否通过促进卵黄物质的积累实现的呢?为了验证这一假设,从细胞水平上观察了红螯光壳螯虾卵黄发生期肝胰腺和卵巢的超微结构,并分析了与营养物质积累相关的基因脂肪酸结合蛋白(Fatty acid-binding proteins FABP)和卵黄蛋白原(vitellogenin Vg) mRNA表达的差异。以确认饲料中的磷脂对红螯光壳螯虾卵黄发生期营养物质积累的影响。结果表明：随着饲料中SL的增加,虾肝胰腺中脂滴的体积增大,数量增多,结构也更加完整,质地更加均匀,卵巢中脂滴和卵黄颗粒的数量也增多,体积增大；同时,当饲料中SL≥2%时,虾肝胰腺和卵巢中FABP mRNA的表达量随饲料中SL的增加而升高(P<0.05), Diet1和Diet2组间无显著差异(P>0.05),但显著低于其他各组(P<0.05)。肝胰腺中VgmRNA的表达量在Diet3组最高,其次是Diet4组,且显著高于其他3组(P<0.05)。以上结果表明：饲料中的SL有利于红螯光壳螯虾卵黄发生期卵巢中营养物质的快速积累。3.饲料中大豆磷脂对红螯光壳螯虾卵黄发生期脂肪代谢及生理生化指标的影响为了进一步探讨磷脂影响红螯光壳螯虾卵黄发生的生理生化机制,对卵黄发生期营养物质的积累途径进行深入了解,观察了红螯光壳螯虾肝胰腺中与脂肪代谢相关的酶以及血淋巴相关指标,并分析了与肝胰腺正常功能相关的转氨酶及抗氧化酶活性的变化。结果显示：Diet5组虾肝胰腺中脂蛋白脂酶的活性显著高于其他4组(P<0.05),Diet4组虾肝胰腺中脂蛋白脂酶的活性与Diet1和Diet3组无显著差异(P>0.05),但显著高于Diet2组(P<0.05)。Diet1组虾肝胰腺的肝脂酶活性与Diet2组无显著差异(P>0.05),但显著低于其他3组(P<0.05)。总脂酶的活性在Diet5组最高,其次是Diet3和Diet4组,最低的是Diet1和Diet2组。当饲料中添加4%SL时,虾血淋巴上清液中甘油三酯的含量与Diet5组无显著差异(P>0.05),但显著高于其他3组(P<0.05)。Diet5组虾血淋巴上清液高密度脂蛋白胆固醇的含量与Diet3和Diet4组无显著差异(P>0.05),但显著高于Diet1和Diet2组(P<0.05)。饲喂6%SL饲料的虾血淋巴上清液中游离脂肪酸的含量与Diet4组无显著差异(P>0.05),但显著高于其他3组(P<0.05)。Diet1和Diet5组虾肝胰腺谷丙转氨酶(ALT)活性显著低于Diet2和Diet4组(P<0.05)。Diet5组虾谷草转氨酶(AST)活性显著低于其他各组(P<0.05)。以上结果表明：饲料中高含量的SL对红螯光壳螯虾肝胰腺和血淋巴上清液中的脂肪代谢有一定的积极作用。同时,虾肝胰腺的抗氧化功能处于一种相对稳定的状态。综合以上结果,饲料中高水平的磷脂对红螯光壳螯虾的卵黄发生具有促进作用。这是通过促进其卵黄发生期营养物质的快速积累以及促进脂肪的代谢吸收而实现的。建议在红螯光壳螯虾亲本培育过程中,在基础饲料中至少补充2%的磷脂以有利于其卵黄发生期营养物质的快速积累和性腺的快速发育。4.红螯光壳螯虾成熟促进因子(MPF)的克隆、表达和定位分析在以上研究基础上,试图从分子水平找到营养调控红螯光壳螯虾卵黄发生的关键因子,选择了与卵黄发生密切相关的成熟促进因子(Maturation-promoting factor MPF)进行了初步探讨。MPF包括催化亚基细胞周期蛋白依赖性激酶2(Cdc2)和调节亚基细胞周期蛋白B (CyclinB),是减数分裂G2/M期转换的关键调节因子,对卵母细胞恢复减数分裂达到成熟起着重要的调节作用。MPF在高等动物已有大量研究,但对其在甲壳动物中的表达特征和功能作用的研究却有限。4.1红螯光壳螯虾Cdc2基因的克隆、序列和表达特征分析以及免疫组化定位的研究本试验克隆得到了红螯光壳螯虾Cdc2基因的cDNA全长序列。该序列全长1769bp,编码299个氨基酸,蛋白分子量约为34.7kDa。实时荧光定量PCR表明,Cdc2mRNA主要在卵巢中表达,且表达量随着卵巢发育的成熟而下降。随着饲料中SL的增加,虾卵巢中Cdc2mRNA的表达量呈现先降低后升高的趋势,其表达量在Diet2, Diet3和Diet4组无显著差异(P>0.05),但显著低于Diet1和Diet5组(P<0.05)。免疫组织化学细胞定位发现,Cdc2蛋白在红螯光壳螯虾卵黄发生过程中存在从细胞质向细胞核的转移。4.2红螯光壳螯虾CyclinB基因的克隆、序列和表达特征分析以及免疫荧光定位的研究红螯光壳螯虾CyclinB cDNA序列全长1779bp,编码401个氨基酸,预测的CyclinB蛋白的分子量和等电点(pI)分别为45.1kDa和8.84。实时荧光定量PCR表明,CyclinB mRNA主要在卵巢中表达,且表达量随着卵巢发育的成熟而下降。随着饲料中SL的增加,虾卵巢中CyclinB mRNA的表达量呈现先降低后升高的趋势,其表达量在Diet2, Diet3和Diet4组无显著差异(P>0.05),但显著低于Diet1和Diet5组(P<0.05)。免疫荧光技术细胞定位发现,CyclinB蛋白在红螯光壳螯虾卵黄发生过程中也存在从细胞质向细胞核的转移。以上结果表明,MPF在红螯光壳螯虾配子形成和性腺发育过程中起着重要作用,饲料中的SL影响卵巢中MPF mRNA的表达。更多还原

【Abstract】 The redclaw crayfish, Cherax quadricarinatus, is a popular commercial crustacean species in several countries around the world because of their potential large size, fast growing, tolerance to a broad range of temperatures and water quality conditions, and large market potential. Since its introduction to China from Australia in1992, it has not formed a large scale cultivation although some scientific researches have done. The main limitation is small-scale larvae supply which was mainly resulted by mature synchronization rate, low rate of spawning and hatching and livability. At present, many studies have focused on the juvenile redclaw’s growth, survival and body composition, but few have evaluated the broodstock reproduction. As broodstock nutrition affects egg quality, embryonic development and postlarval quality in crustaceans, an evaluation of ovarian maturation will be helpful in understanding the nutrition requirements of the female redclaw during vitellogenesis. In this study, we tried to understand the mechanism of dietary lecithin affecting Cherax quadricarinatus gonadal development based on nutrition, biochemistry, electron microscope and molecular biology methods. The data obtained would provide valuable information to a better understanding on vitellogenesis of crustaceans. It is also helpful to redclaw’s large-scale commercial aquacculture.1. Effects of dietary soybean lecithin on growth and biochemical changes of hepatopancreas and ovary in the female redclaw crayfish, Cherax quadricarinatus.The effects of increasing levels of dietary de-oiled soybean lecithin (SL) on growth and biochemical changes of hepatopancreas and ovary during gonadal development of female redclaw crayfish, Cherax quadricarinatus, was investigated. Five practical formulated diets were supplemented with0%(Diet1),1%(Diet2),2%(Diet3),4%(Diet4) and6%(Diet5) SL. Crayfish (initial weight:25.64±1.53g, eight shrimp per tank) were fed each diet in four replicates for8weeks. Shrimp survival and relative weight gain were not significantly different among the five treatments(P>0.05). Crayfish that were fed diets containing≥2%SL had a significantly higher gonadosomatic index (GSI) than those fed with Diets1and2(P <0.05), while hepatosomatic index (HSI) showed a decreasing trend (P>0.05) with increasing dietary SL. The fatty acid composition of the hepatopancreas is largely due to the dietary composition. Higher dietary SL yielded a higher percentage of ovarian polyunsaturated and highly unsaturated fatty acids (P<0.05). Furthermore, the higher content of linoleic acid and alpha linolenic acid in diets may be more important for ovarian development in C. quadricarinatus, as these originate from the hepatopancreas. Our results suggest that dietary SL has a positive effect on vitellogenesis in C. quadricarinatus broodstock.2. Effects of dietary soybean lecithin on tissue ultrastructure, fatty acid-binding proteins and vitellogenin mRNA expressions in the female Cherax quadricarinatus.Did dietary SL promote vitellogenesis of C. quadricarinatus by promoting yolk nutrients accumulation? For the hypothesis, hepatopancreas and ovary ultrastructure, fatty acid-binding proteins (FABP) and vitellogenin (Vg) mRNA expressions of female redclaw crayfish, Cherax quadricarinatus, was investigated. With increasing percentages of dietary SL, the hepatopancreatic lipid droplets numbers and sizes increased and the structure and texture was more complete and distributed. The ovarian lipid droplets and yolk granules numbers and sizes increased. When dietary SL≥2%, shrimp hepatopancreatic and ovarian FABP mRNA expression increased with increasing dietary SL level (P<0.05). There were not significantly different between Diet1and Diet2for shrimp hepatopancreatic and ovarian FABP mRNA expressions (P>0.05), but they were lower than that of other treatments (P<0.05). The hepatopancreatic Vg mRNA expression was the highest in crayfish that received Diet3. Our results suggest that dietary SL can enhance ovarian nutrient accumulation in C. quadricarinatus broodstock.3. Effects of dietary soybean lecithin on lipid metabolic and physiological and biochemical index in the female Cherax quadricarinatus.In order to further understand physiological and biochemical mechanism of dietary lecithin affecting vitellogenesis and way of nutrients accumulation of Cherax quadricarinatus, we evaluate the effects of lipid metabolism and hepatopancreatic antioxidant capacity of shrimp fed diets containing increasing levels of SL(0%(Diet1),1%(Diet2),2%(Diet3),4%(Diet4)和6%(Diet5)). The hepatopancreatic lipid metabolism enzyme and hemolymph supernatant index were analyzed. Hepatopancreatic lipoprotein lipase (LPL) of shrimp in Diet5was higher than other four treatments(P<0.05). LPL in Diet4was not significantly different with that in Diet land Diet3(P>0.05), but it was higher than diet2(P<0.05). Shrimp hepatopancreatic hepatic lipase (HL) in diet1was not significantly different with Diet2(P>0.05), but it was higher than other three treatments(P<0.05). Total lipase was highest in diet5, following diet3and diet4, the lowest was diet1and diet2. When fed4%SL feed, shrimp hemolymph supernatant triglyceride was not significantly different to diet5(P>0.05), but significantly higher than other treatments (P<0.05). High density lipoprotein cholesterol in diet5was not significantly different to diet3and diet4(P>0.05), but significantly higher than diet1and diet2(P<0.05). When fed6%SL feed, shrimp hemolymph supernatant free fatty acid was not significantly different to diet4(P>0.05), but significantly higher than other treatments (P<0.05). Shrimp hepatopancreatic alanine aminotransaminase (ALT) in diet1and diet5were significantly lower than that in diet2and diet4(P<0.05). Aspartate aminotransaminase (AST) in diet5was significantly higher than other four treatments (P<0.05). Our results suggest that high dietary SL has a positive effect on hepatopancreatic and hemolymph supernatant lipid metabolism. When fed high SL feed, shrimp was still in steady-state of hepatopancreatic antioxidant system.Over all above results, dietary SL has a positive effect on vitellogenesis in C. quadricarinatus broodstock, which is accomplished by promoting rapid accumulation of nutrients and metabolism and absorption of lipid during vitellogenesis period. We suggest that at least2%SL should be supplemented in broodstock diets to enhance ovarian nutrient accumulation and ovary development during aquaculture.4. Molecular cloning, characterization expression and localization analysis of MPF in the red claw crayfish (Cherax quadricarinatus).Based on the results above, we further investigated the maturation-promoting factor (MPF), a central factor for inducing the meiotic maturation of oocytes, to try to understand the molecular nutrition mechanism. MPF has been studied in a wide variety of animal species; however, its expression in crustaceans is poorly characterized.4.1Characterization of Cdc2kinase in the red claw crayfish (Cherax quadricarinatus):Evidence for its role in regulating oogenesis.Cdc2kinase is a catalytic subunit of MPF, a central factor for inducing the meiotic maturation of oocytes. In this study, a complete cDNA sequence of Cdc2kinase was cloned from the red claw crayfish, Cherax quadricarinatus, and its spatiotemporal expression profiles were analyzed. The Cdc2cDNA (1769bp) encodes for a299amino acids protein with a calculated molecular weight of34.7kDa. Quantitative real-time PCR demonstrated that Cdc2mRNA was expressed mainly in the ovary tissue and the expression decreased as the ovaries developed. With increasing dietary SL, shrimp ovarian Cdc2mRNA expression decreased firstly and then increased again. The expression was not significantly different among diet2, diet 3and diet4(P>0.05), but significantly lower than diet1and diet5(P<0.05). Immunohistochemistry analysis revealed that the Cdc2protein relocated from the cytoplasm to the nucleus during oogenesis.4.2Characterization of CyclinB in the red claw crayfish (Cherax quadricarinatus):Evidence for its role in regulating oogenesis.CyclinB is a regulatory subunit of MPF. The CyclinB cDNA (1779bp) of Cherax quadricarinatus encodes for a401amino acids protein with a calculated molecular weight and isoelectric point of34.7kDa and8.84. Quantitative real-time PCR demonstrated that CyclinB mRNA was expressed mainly in the ovary tissue and the expression decreased as the ovaries developed. With increasing dietary SL, shrimp ovarian CyclinB mRNA expression decreased firstly and then increased again. The expression was not significantly different among diet2, diet3and diet4(P>0.05), but significantly lower than diet1and diet5(P<0.05). Immunohistochemistry analysis revealed that the CyclinB protein also relocated from the cytoplasm to the nucleus during oogenesis. These findings suggest that MPF may play an important role in the gametogenesis and gonad development in C. quadricarinatus. And dietary SL influenced shrimp ovarian MPF mRNA expression.更多还原