【作者】 李莉；

【导师】 李琪；

【作者基本信息】 中国海洋大学 ， 遗传学， 2009， 博士

【摘要】 刺参(Apostichopus japonicus)是我国重要的经济养殖种类,具有极高的经济价值。本文分别研究了我国青刺参和日本红刺参苗种培育的生物学特征及杂交育种。主要结论如下:1温度和盐度对青刺参幼虫生长和存活的影响在4个温度梯度(18,21,24和27℃)和8个盐度梯度(10,15,20,25,30,35,40和45‰)条件下,分别研究了温度和盐度对刺参摄食前及摄食后幼虫存活和生长发育的影响。结果表明:(1)摄食前和摄食后幼虫生长和存活的适宜温度为21-24℃;温度超过其适宜范围时,幼虫的存活率显著下降。(2)60 h内,受精卵能够正常发育至初耳幼虫的盐度范围为20-35‰;盐度过高或过低时,幼虫发育畸形或死亡;幼虫在自然海水盐度(30‰)时的存活率、变态率和生长速度皆最高。2不同附着基与环境因子对青刺参幼虫附着变态的影响分别对不同附着基、微生物粘膜以及组织提取液对刺参幼虫附着变态的影响进行了试验研究。附着基分别采用透明波纹板、塑料薄膜和网片。结果表明:不同附着基间刺参幼虫附着变态效果差异极显著(P < 0.05),透明波纹板是刺参适宜附着基,幼虫的变态率最高。另外,微生物粘膜可以显著的促进幼虫的附着变态(P < 0.05),富含硅藻的粘膜幼虫变态率最高,其次是富含EM益生菌的粘膜和普通微生物粘膜,未经处理的附着基幼虫变态率最低。同种组织提取液也对幼虫的附着变态起到显著的诱导作用(P < 0.05)。分析了温度、盐度和光照等环境因子对刺参幼虫附着变态的影响。实验分别设置了4个温度梯度(18,21,24和27℃),4个温度盐度(20,25,30和35‰),以及3种光照(蓝色、绿色和黑色)。结果表明,刺参附着变态的适宜温度为18-21℃,且幼虫变态率随温度升高而增大;当温度超过适宜范围(> 24℃)时,幼虫的变态率随着温度升高而减小;温度对幼虫附着变态的影响极为显著(P < 0.001)。幼虫附着变态的适宜盐度为30‰;盐度的升高或降低对幼虫附着变态的影响极显著(P < 0.001)。光照对幼虫附着变态的影响显著(P < 0.05),较弱的光照更适宜刺参幼虫的附着变态。3不同营养添加剂对青刺参生长的影响对螺旋藻、EM益生菌、低聚糖和多维等添加剂在刺参苗种生长中的作用进行初步探索。结果表明:螺旋藻、海参多维和低聚糖均能促进刺参苗种的生长,实验组苗种的质量增长率显著高于空白对照组;EM益生菌的投喂效果低于空白对照组,其最适添加量有待进一步的研究。投喂各种不同的添加剂对2月龄刺参苗种的脏壁比没有明显影响,均在0.13-0.15之间。4密度、温度和盐度对日本红刺参幼虫生长和存活的影响分别分析了密度、温度和盐度对日本红刺参摄食前和摄食后幼虫生长和存活的影响,为大规模人工育苗确立红刺参苗种适宜生长的环境条件。结果表明:摄食前幼虫在育成密度为0.5-1.0个/ml时日生长、出苗量、育成成活率最高;摄食后幼虫的适宜育成密度为0.1-0.2个/ml;随着育成密度增加,幼虫存活率和生长速度呈下降趋势。摄食前和摄食后幼虫在21-24℃时存活率最高,生长最快;温度过高,促进幼虫的生长,但存活显著下降;温度过低,幼虫的存活和生长均显著下降。幼虫适宜的盐度为30‰;随着盐度的升高和降低,幼虫生长和存活率下降,畸形和死亡率上升。5日本红刺参盐度耐受性的研究将日本红刺参的稚参分别在3个温度梯度(21℃,24℃和27℃)和3个盐度梯度(32‰,27‰和22‰)组成的9个温、盐度结合组中暂养20 d,然后以渐变的方式对刺参的盐度耐受性进行研究。结果表明:稚参的SSMax、SSMin、50% CSMin和50% CSMax值均与暂养盐度成正相关,与暂养温度成负相关。双因子方差分析显示,温度和盐度以及温盐度的共同作用都对稚参的存活影响显著(P≤0.001)。6刺参杂交子一代的微卫星和AFLP分析本研究利用中国当地刺参(♀)和日本红刺参(♂)进行了杂交试验,并使用用微卫星和AFLP标记分析了杂交子一代的遗传特性。微卫星标记对两亲本及其杂交子代的分析结果表明,杂交子代的等位基因均来自两亲本,为真正的杂交种。8对AFLP引物共扩增出375个位点,其中多态位点265个,多态位点比例69.8%;238个位点在子代中发生分离,符合孟德尔分离规律的位点175个,其中母本69个,父本66个;偏离孟德尔分离的位点43个;异常分离位点20个。子一代与母本间的遗传距离为0.0391,小于其与父本之间的遗传距离(0.0999),表明杂交子代在遗传上与母本较近。更多还原

【Abstract】 The sea cucumber Apostichopus japonicus Selenka is an importantly commercial species in China, with good market value. In the present study, the culture biology of the green and red sea cucumber and the hybridization of the two color variants have been investigated. The main results are following:1 Effects of temperature and salinity on larval growth, survival and development of the green race, A. japonicusIn this study, two separate experiments were conducted to investigate the effects of temperature and salinity on survival and growth of Apostichopus japonicus larvae before and after feeding. 4 different levels of temperature (18, 21, 24 and 27°C) and 8 different levels of salinity (10, 15, 20, 25, 30, 35, 40 and 45‰) were investigated in A. japonicus larvae. The optimal temperature for both non-feeding and feeding larvae of sea cucumber was about 21 to 24°C. And the higher temperature (27°C) increased larval growth but reduced the survival in the culture. The production of normal early auricularia larvae from fertilized eggs were completed after 60 h at salinities of 20-35‰, but not at lower or higher salinity levels. And the best salinity was around 30‰for the normal development and transformation of A. japonicus larvae. The results obtained in this study demonstrate that water temperature of 21-24°C and salinity of 30‰were the most suitable conditions for the early development of A. japonicus.2 Effects of different substrates and environmental factors on larvae settlement in the green raceIn the present study, the effect of various substrata on the settlement of sea cucumber, A. japonicus, was evaluated. The settlement of the larvae in the experiments with three different substrata was compared and showed that polyethylene plastic sheet attracted significantly more larvae (P < 0.05) than plastic film and monofilament nylon. In addition, the highest setting rate was achieved for the plastic sheet with diatom-based films, followed by effective microorganisms (EM)-based films and natural microbial films, and the least recorded was on the plastic sheet without biofilms. There was a significant difference (P < 0.05) in the mean setting rates of the treatments, except between the diatom-based films and the EM-based films. Plastic sheets also appeared attractive to setting larvae when coated with tissue extracts of the same species. The number of settled larvae on substrates with tissue extracts was significantly greater (P < 0.05) than that on the control.Experiments were conducted to determine the effects of temperature, salinity and illumination on larval settlement of sea cucumber. The larvae were reared at four levels of temperature (18, 21, 24 and 27°C), four levels of salinity (20, 25, 30, and 35‰), and three levels of illumination (blue, green and black). The optimal temperature for larval settlement was 18-21°C, and the larval setting rate increased directly with temperature. However, when the temperature was over the optimal range (> 24°C), the larval setting rate was inversely related to temperature. There was a significant difference of larval settlement at different temperatures (P < 0.001). The optimal salinity for larval settlement was 30‰. The effects of higher or lower salinities on larval setting rate were significant (P < 0.001). There was a difference (P < 0.05) in the mean setting rates of the different levels of light. Larval settlement preference was associated with light avoidance behaviour, and the darkness was optimum for sea cucumber larval settlement.3 Effects of different nutritional additive on growth of the green race juvenileExperiments were conducted in culture tanks to investigate the effects of four different nutritional additive (Spirulina, Effective Microorganisms, Oligosaccharides, and Multivitamins) on juvenile A. janponicus. The results showed that the growth was significantly affected by spirulina, oligosaccharides and multivitamins, the weight growth rates of which were all higher than the control. There was no significant effect of EM on the growth of juvenile, compared to the control. The added level of EM needed advanced study. Additionally, the effects of different nutritional additives on the viscera body wall ratio of two-month old juveniles were not significantly different (0.13-0.15).4 Effects of stocking density, temperature and salinity on larval survival and growth of red A. japonicus The red race of the sea cucumber A. japonicus was introduced into China from Japan for large-scale seeding production because of its economical value. This paper reports the effects of stocking density, temperature and salinity on the survival and growth of early larvae before and after feeding, in order to establish the conditions for optimal larval growth. To maximize the yield per unit of space, densities of 0.5-1 larvae/ml is recommended for the non-feeding larvae, while 0.1-0.2 larvae/ml is appropriated for the feeding larvae. Higher survival and growth values were obtained for both the non-feeding and feeding larvae at temperature ranged from 21 to 24°C. And the larvae reared at salinity of 30‰always showed the maximum of growth and survival. Based on the results, a temperature from 21 to 24°C and a salinity of 30‰are optimum for the early development of the red A. japonicus.5 Salinity tolerance of red A. japonicus juvenile at different salinity and temperature levelsThe juveniles of red race sea cucumber acclimated in different environmental conditions (21, 24 and 27°C combined with 22, 27 and 32‰) were tested for salinity tolerance by increasing and decreasing salinity at a rate of 2‰. The results showed that the values of SSMax (Survial salinity maximum), SSMin (Survival salinity minimum), 50% CSMin (Critical salinity minimum) and 50% CSMax (Critical salinity maximum) all increased directly with salinity, but were inversely related to temperature. In the present study, an analysis of two-way ANOVA showed that salinity and temperature had a significant effect on survival of juveniles (P≤0.001).6 Microsatellite and AFLP analysis of the hybridization of sea cucumber Apostichopus japonicusThe hybrids between two colour variants of sea cucumber Apostichopus japonicus (the green♀×the red♂) were produced in this study, and the genetic relationship between the F1 progenies and their parents were examined using microsatellite and AFLP analyses. The results of microsatellite analysis confirmed that the F1 progenies were the hybrids between the two colour variants of sea cucumber. As a result of AFLP analysis, eight primer combinations generated 375 loci, among which 265 were polymorphic in F1 generation, accounting for 69.8%. Among the 238 segregating loci, 175 loci segregated in Mendelian model, including 69 maternal specific loci, 66 paternal specific loci and 40 mutual loci. Forty-one loci deviated from Mendelian ratios and 20 segregated in abnormal model. The genetic distance was 0.0391 between the F1 progenies and female parent, and 0.0999 between the F1 progenies and male parent, respectively, indicating that the hybrids were genetically closer to their maternal broodstock.更多还原