【作者】 朱葆华；

【导师】 王广策；

【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， 2005， 博士

【摘要】 海洋无脊椎动物与甲藻之间的共生关系在海洋环境中普遍存在,这些甲藻称之为虫黄藻,属于共生甲藻属(Symbiodinium)。共生藻吸收无脊椎动物代谢产生的二氧化碳、磷酸盐、硝酸盐等,并转化为共生体所需要的营养。因此,共生藻的存在是共生体进行正常生理活动所必不可少的。 本研究以几种海洋无脊椎动物-共生藻的共生体为研究对象,从细胞水平、个体水平以及分子水平对共生规律进行了一些探讨,得到如下一些结果。 通过石蜡组织切片和扫描电镜组织切片观察了共生藻在 3 种海葵触手组织中的分布,通过透射电镜研究了共生藻在宿主细胞中的定位。结果表明,共生藻主要存在于触手组织的内胚层,被宿主细胞的空泡包围。本研究至少可以对共生藻在宿主体内的生活史进行一定的补充,即共生藻在分裂的过程中,首先形成新的周质膜把新分裂形成的细胞包围起来,然后再逐渐被宿主细胞的空泡分开,最后一个空泡只包含一个共生藻细胞。 有关共生藻的研究,离体培养是进一步研究的基础和非常关键的环节,也是一个难点。本实验以 3 种海葵的共生藻为实验材料,探讨了共生藻的体外培养。研究结果表明:共生藻在体外较难培养,培养 10 小时后,共生藻的表面就会出现小的空洞,随着时间的延长,表面的空洞逐渐增大,并出现细胞内容物外溢的现象。 以不同饥饿时间的地毯海葵为研究对象,研究了饥饿时间对共生藻的影响。结果表明,随着海葵共生体饥饿时间的延长,共生藻的细胞数目显著降低,叶绿素(叶绿素 a 与叶绿素 c)含量也随之降低,最大量子产率有升高的趋势,而细胞的大小没有受到影响。共生藻的超微结构也有一些变化,饥饿 3 天的海葵含有正在分裂的细胞,其共生藻含有较多数目的线粒体,而饥饿 45 天与饥饿 280 天海葵体内的共生藻出现了较多的脂肪颗粒。 克隆了巨型辐花海葵(Radianthus macrodactylus)与地毯海葵(Stichodactyla mertensii)共生藻的 ITS 序列,并对序列进行了分析。结果表明,这 2 种共生藻的 ITS1 均为 223 bp,5.8 S 均为 159 bp,ITS2 均为 191 bp,ITS 序列全长(包括 5.8 S)为 573 bp。序列分析结果表明,2 种共生藻带有 5.8 S 的 ITS 片段仅有更多还原

【Abstract】 Symbiotic associations between invertebrates and dinoflagellates are a common occurrence in marine environments. These dinoflagellates, collectively known as zooxanthellae, predominately belong to Symbiodinium. These symbiotic algae absorb carbon dioxide, phosphate and nitrate produced by invertebrates and transform them into nutrition necessary to symbiont (symbiotic algae and invertebrate). Therefore, it is absolutely necessary for symbiont to have their symbiotic algae. To explore the mechanism of symbiosis, some algal-invertebrate associations were studied from several aspects, including cell level, individual level and molecular level. Distribution of symbiotic algae in the tissue of three kinds of anemone was studied by microscope and scanning electron microscope (SEM). Localization of these symbiotic algae in the cell of the hosts was also confirmed using transmission electron microscope (TEM). The results showed that symbiotic algae were found predominantly in the endoderm of the tentacles and were located within vacuoles in host cells. Culturing symbiotic algae in some medium successfully is cornerstone and has great difficulty to the study of symbiotic algae. Symbiotic algae from three kinds of anemone were cultivated in medium in this study. The results showed that it was very difficult to culture symbiotic algae in artificial medium. Some small holes occurred on the surface of symbiotic algae after being cultivated for 10 h. Holes were enlarged and some cell contents were discarded with extending cultivating time. Effects of starvation time on symbiotic algae in different anemone Stichodactyla mertensii at different starved time were studied. The results showed that the number of symbiotic algae and the content of chlorophyll a (c) significantly decreased with prolonging anemone-starved time, while maximum quantum efficiency (Fv/Fm) of symbiotic algae had a tendency to rise. Cell size of symbiotic algae was not affected by starved time of animals. Some changes in the ultrastructure of symbiotic algae occurred. Symbiotic algae from anemone starved for 3 days contained some dividing cells and had more mitochondria than that from starved anemones for 45 days and 280 days. The latter algae contained some lipid grains. ITS (internal transcribed spacer) sequence of symbiotic algae from Radianthus macrodactylus and Stichodactyla mertensii were cloned and analysed. The results showed the full length of the two ITSes from both the symbiotic algae were 573 bp (including 5.8 S). ITS1, 5.8 S, ITS2 were 223 bp, 159 bp,191bp respectively. The analysis of the sequence indicated that the similarity was up to 99.13% of the two ITS sequence (including 5.8 S) from both symbiotic algae. The phylogenetic tree constructed with 5.8 S rDNA using Neighbor-joining (NJ) method showed that the two symbiotic algae belonged to clade F. But they might belong to clade C in the phylogenetic tree using ITS+5.8S and 5.8S+ITS2. However, the close phylogenetic positions between two symbiotic algae was not changed according to the phylogenetic tree with different ITS regions because clade C and clade F are sister groups. Effects of temperature, hypoxia, ammonia and nitrate on symbiotic associations between symbiotic algae and three coral species were studied through examination of morphology and the measurement of the number of symbiotic algae of three coral species Acropora nobilis, Palythoa sp. and Alveopora verrilliana. Results showed that increase in temperature and decrease in dissolved oxygen could lead to increasing number of symbiotic algae and the breakdown of symbiotic associations. In addition, the concentration of 0.001 mmol/L ammonia or nitrate could increase significantly the expulsion of the symbiotic algae of the three coral species. Except for Acropora nobilis, the numbers of symbiotic algae of other two corals did not significantly increase with the increasing concentration of ammonia and nitrate. Furthermore, different hosts have different stress susceptibilities on symbiotic associations.更多还原