【作者】 张成松；

【导师】 相建海； 李富花；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2009， 博士

【摘要】 虾蟹类甲壳动物养殖业是我国重要的经济产业之一,为解决沿海及内陆农民的就业和经济增收做出了巨大贡献,产生了巨大的社会和经济效益。但近年来由于种质退化、环境恶化和病害猖獗等原因,虾蟹养殖业的可持续发展受到了严重制约,因此开展虾蟹类的遗传改良势在必行。研究表明,水产动物的多倍体育种和单性化养殖在提高产量、改善品质等方面具有重要的促进作用。本论文选取中国明对虾、凡纳滨对虾和中华绒螯蟹这3个我国重要的养殖品种进行了多倍体和雌性化控制的研究,优化了中国明对虾和中华绒螯蟹的多倍体诱导方法及凡纳滨对虾雌性化控制的关键参数,并在此基础上研究了三倍体中国明对虾在早期生长阶段的生产性状和生物能量学特征。在中国明对虾受精卵第一次成熟分裂以前,利用热休克的方法抑制第一极体释放可以获得高比例的三倍体,实验未发现有类似于贝类非整倍体及四倍体存在的现象;热休克显著降低了中国明对虾的变态存活率,而倍性本身对其影响不明显;倍性操作显著影响了中国明对虾的体形特征,三倍体对虾体形粗短而二倍体对虾体形修长;根据部分形态学指标发展了中国明对虾倍性判别公式,其判别准确率在90%以上。利用热休克方法抑制第二极体释放获得高比例的中国明对虾三倍体,并在实验室条件下对三倍体和二倍体对虾在幼虾阶段的生长性状进行了比较。结果表明:倍性操作对中国明对虾幼虾阶段的急性盐度突变的适应能力及摄食率无明显影响,但其对三倍体中国明对虾的养殖成活率、生长率、饵料转化效率及蜕皮周期有显著影响(P<0.05);二倍体对虾的最适生长盐度为20‰,而三倍体对虾为30‰,在幼虾阶段,三倍体中国明对虾未表现出明显的生长优势。在实验室条件下对三倍体和二倍体中国明对虾在性腺发育之前和性腺发育早期的生物能量学特征进行了比较研究,结果表明倍性操作对中国明对虾的基础代谢水平和能量收支模式有显著的影响。在性腺发育以前,在实验盐度范围(15~35‰)和温度范围内(26~32℃),三倍体对虾的耗氧率显著高于二倍体对照(P<0.05),而在某些盐度或温度梯度下,三倍体对虾的排氨率也明显高于二倍体对照(P<0.05),但三倍体对虾分配于生长的能量份额明显低于二倍体(P<0.05);在性腺发育早期,三倍体对虾的耗氧率及排氨率仍明显高于二倍体对虾(P<0.05),其分配于生长的能量份额较高,但与二倍体对虾无明显差异(P>0.05),本研究为三倍体中国明对虾早期生长性状的解释提供了生物能量学基础。对中华绒螯蟹脱落及附着受精卵在产出后的形态学指标进行了观察测量,并基于形态学的观察,优化了河蟹三倍体诱导的处理条件。结果表明:河蟹受精卵在产出后经历了一个体积膨胀的过程,受精膜经历了形成举起、举起达最高再降低的过程,而卵黄团的位置也随发育时间而改变;初步确定了一个不受温度、遗传背景等影响的可用于大批量生产河蟹三倍体的起始处理时刻的形态学标记-受精膜举起,大大提高了中华绒螯蟹三倍体诱导效率。首次尝试并优化了凡纳滨对虾雌性化控制的诱导方法,结果表明:凡纳滨对虾受精卵对温度的耐受力随发育而提高;在原肠中后期,热休克处理(39℃处理3min)可以显著提高诱导群体的雌性比例。更多还原

【Abstract】 Crustacean cultivation (especially to shrimp and crab), which has contributed largely to the employment of rural population and the augment of rural incomes, is one of the economically important industries in China, it has brought us huge social and economic benefit. Unfortunately, for the reasons of serious diseases, idioplasmic degeneration and environmental deterioration, the development of culture in shrimps and crabs encountered prodigious handicap in China in recently years. So the genetic improvement to the crustacean species is imperative. Previous researches on polyploid breeding and mono-sex culture of aquatic animal have been proved to be successful in increasing yield and improving quality. In the present thesis, the Chinese shrimp (Fenneropenaeus chinensis), the Pacific white shrimp (Litopenaeus vannamei) and the Chinese mitten crab (Eriocheir japonica sinensis) were selected as the experimental animals. Heat shock or potassium chloride (KCl) was employed to induce polyploidy in F. chinensis or E. japonica sinensis, and the feminization control was carried out in L. vannamei by heat shock.Heat shock was employed to inhibit the releasing of the first polar body to produce meiosisⅠtriploid F. chinensis. At 15℃, heat shock (temperature, 29-31℃; duration, 8-10min ) successfully induced MⅠtriploids when treated at 7min post spawning, and the highest triploid rate is about 100 percent. No aneuploid and tetraploid were observed in treated samples by inhibition of the 1st polar body. Ploidy itself did not affect shrimp larvae survival during metamorphosis, but heat shock significantly decreased the survival rate. Ploidy manipulation significantly affected the morphological characteristics of F. chinensis, the body shape of triploid shrimp is dumpy, but the diploid shrimp is slender. Based on morphological parameters, discriminating formulae for triploid and diploid shrimp at grow-out stage were developed and could be used to distinguish triploids from diploids.Triploid Chinese shrimp Fenneropenaeus chinensis were reared up by heat shock inducement to inhibit meiosisⅡ. The growth performances in full-sib diploid and triploid F. chinensis were compared to evaluate the effect of ploidy manipulation at juvenile stage under laboratory conditions. It showed that there was no significant difference (P>0.05) in tolerance observed in triploid and diploid shrimp due to abrupt salinity changes. The lethal salinity for 50% of the individuals in 96h at 23-25℃was about 2‰in both triploids and diploids. Ploidy significantly influenced (P<0.05) the survival rate, special growth rate, feed conversion efficiency and intermolt period of F. chinensis, but there were no difference of the feeding rate between the two ploidy levels. Based on the survival and growth data, the optimum salinity for the culture of diploid F. chinensis should be 20‰and for triploids it should be 30‰. At the early stage during culture, triploid F. chinensis juvenile showed no growth superiority on their diploid controls .The effects of ploidy manipulation on bioenergetics of F. chinensis were studied under laboratory conditions, and the bioenergetics’bases to explain the different growth performance during culture period were proved up. At immature stage, triploid shrimp exhibited higher oxygen consumption rate than the diploids at the range of acclimation salinities (15-35ppt) and temperatures (26-32°C), and higher ammonia-N excretion rate at some salinity level (25 and 35ppt) and temperature level (32°C). Triploid shrimp have lower energy allot for growth than that of the diploids (P<0.05). The tendencies of environmental factors on standard metabolic rate were similar between the two ploidy levels. At the early stage of gonad development, there was no difference of energy budget between triploid and diploid shrimp (P>0.05), but the triploids exhibited higher metabolic rate (P<0.05). This is the first report of bioenergetics in triploid shrimp and it likely explain the prior experimental results which with difference of growth performances between triploid and diploid shrimp.Based on morphological observation of deciduous and normal attached fertilized eggs of E. japonica sinensis, a potential morphological marker (the apparition of the plasma membrane and apparition of maximum distance between the plasma membrane and yolk mass) which can be used as the optimum starting time shocked by KCl was determined. This is the first report of morphological dynamics of early development of fertilized eggs from Chinese mitten crab, and later repeated experiments indicated that the morphological marker can be used as an indicator for highly efficient triploid induction with KCl in this crab species.To mono-sex culture, heat shock was employed to feminize the L. vannamei at different stages of embryonic development. It showed that the thermotolerance of shrimp embryos raised as the development advanced, at middle and later stages of gastrulae, heat shock (temperature, 39℃; duration, 3min) significantly increased the female ratio in the treated populations. This is the first report of feminization in L. vannamei treated with heat shock.更多还原