Axenic Cultivation Of Symbiodinium Sp. From Several Coelenterates And Investigation For Their Physiology

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.