Biological Experiment of Zooxanthellae
This research is designed to explore in vitro Zooxanthellae cell cultures with addition of nutritional elements such as P, Fe, N and VB12 to test their tolerance of heat shock as environmental stress. The aim is to understand which nutrients will enhance the resistance of Zooxanthellae against temperature elevation. The study is of critical importance for the health and metabolic balance of marine coral microecosystem under global climate change.
Firstly, we cultivate Zooxanthellae in vitro with ordinary growth condition to obtain the baseline of the cell cultures. Secondly, various concentrations of certain elements are used to determine which nutrients are able to enhance the Zooxanthellae resistance to heat stress. Thirdly, optimal concentrations for selected nutrients are applied for Zooxanthellae to strengthen their defense against stress. Finally, chlorophyll fluorescence is measured to understand the viability of Zooxanthellae cells by their photosynthetic efficiency with given nutrient concentrations during heat shock. With the above experimental procedure, we are able to explain how the supply of certain nutrients will prevent Zooxanthellae from bleaching and the what concentrations of nutrients are needed to enhance the health of coral microecosystem.
1. Ordinary growth of Zooxanthellae and Pseudomonas denitrificans
We chose three clades of Zooxanthellae, clade B1, clade C92 and clade E1, in our study. f/2 medium is used for ordinary growth of Zooxanthellae. It is found that the growth rate of Zooxanthellae clade E1 was higher than clade B1 and clade C92. ,The growth curve for clade E1 is shown in Figure 1 .
Additionally, we have grown the bacterium Pseudomonas denitrificans (see Figure 2 for its growth curve) . It is known that Pseudomonas denitrificans is capable of producing vitamin B12 (VB12) in bioindustry. Together with nitrogen fixation and phosphorus dissolving property. Therefore, this strain was selected for our project.
2. Determination of nutrients effective for health of Zooxanthellae under heat shock
After literature survey, we chose four nutritional elements, N, Fe, P and VB12, that are critical to healthy growth of Zooxanthellae for our experiments. N, Fe, P and VB12 with concentrations of 1, 10 and 100 times that of the control group were added to the Zooxanthellae clade E1 cell cultures and then the Fv/Fm value was measured to find the optimal concentration. After measurement, Zooxanthellae will be cultured in a new medium.After the experiment, we drew primary solution of the best concentration of each element for resisting coral bleaching. According the collected data, the approximate best concentration of P was between 1x and 10x; the best concentrations of Fe was between 10x and 100x and that of VB12 was between 1x and 10x. However, we had not discovered the approximate best concentration of N from the data.
It was also found that Fe was relatively less effective. N, P and VB12 were selected for further investigation.
3. Determination of optimal nutrient concentrations for Zooxanthellae against heat shock
In the further experiment, we still took the two groups of Zooxanthellae B1 and C92 and added N, P and VB12 with concentrations of 1, 10 and 100 times respectively. In one of the C92 corals, we particularly added VB12 with concentrations of 20, 40, 60 and 80 beside the concentrations of 1, 10 and 100. Then in the first three days of the experiment, the samples were taken out of the light for 5 minutes in every measurement and their Fv/Fm values were measured. On the third day, Zooxanthellae was bleached, and the Fv/Fm values of the samples were measured every two days after the albination.
From the data we gathered, we discovered that before bleaching, the best concentration of VB12 was between 60x and 80x; after alination, the turning point kept at the point of 80x and thus, the best concentration of VB12 was 80x. The best concentration of P was 10x while the data still did not reflect the best concentration of N.
4. Measurement of chlorophyll fluorescence for viability of Zooxanthellae cells with given nutrition.
Chlorophyll fluorescence can be used as a fast, sensitive and nondestructive method to investigate the effects of various stress factors on photosynthesis of micro-algae. The measurements involve abundant photosynthetic and viability information. The Fv/Fm value reflects the potential maximum photosynthetic capacity of algae under different growth conditions. Photosynthetic efficiency is the most important indicator of the health status of Zooxanthellae, just like the human electrocardiogram. By measuring the photosynthetic efficiency of Zooxanthellae, we can observe whether the photosynthetic system of Zooxanthellae is healthy and whether the addition of P, N and VB12 has a protective effect on Zooxanthellae.
Zooxanthellae cells appear brown in color, and are usually in coccoid forms surrounded by a cellulose-based cell wall (Fig. 9A). Free-living zooxanthellae were observed using fluorescence microscopy. Little red auto-fluorescence was detected, representing the cellular zooxanthellae chlorophyll (Fig. 9B). During the thermal stress, some of the zooxanthellae were bleached and degraded during the bleaching period (Fig. 9C). The chloroplast content in the zooxanthellae decreased, resulting in a more light brown color than the freshly isolated cells. The bleached cells clumped together and the auto-fluorescence became significantly weaker (Fig. 9D). The morphological observations showed that the bleached cell was bleached and the cell wall was broken, which were similar to the previously reported changes in the host.
They inhabit a specialized host vacuole called a symbiosome. Fluorescence microscope observations of healthy Sarcophyton.sp tentacles revealed the presence of abundant Symbiodiniaceae cells in the gastrodermal layer (Fig. 10A). In contrast, we have shown that the thermal stress caused the bleaching of Sarcophyton.sp tentacles due to the loss of symbiodiniaceae cells, leading to the bleaching of the coral (Fig. 10B). During bleaching, the amount of green fluorescence in the host body and tentacles increased significantly (Fig. 10C). This study also revealed the occurrence of the reduction in gastrodermal Symbiodiniaceae densities (Fig. 10D).
Bourne David G,Morrow Kathleen M,Webster Nicole S. Insights into the Coral Microbiome:Underpinning the Health and Resilience of Reef Ecosystems.[J]. Annual review of microbiology, 2016,70.
David G. Bourne, Kathleen M. Morrow, Nicole S. Webster. Insights into the Coral Microbiome: Underpinning the Health and Resilience of Reef Ecosystems. 2016, 70:317-340.
 Denis Allemand. WALTER M. GOLDBERG. 2013. The Biology of Reefs and Reef Organisms. 2014, 23(3):71-72.