Ex situ cultivation of the soft coral Sinularia flexibilis for biotechnological exploration

Many of the marine sessile invertebrates such as soft corals produce toxins that help protect the coral from competitors and predators. These toxins are of medical importance (e.g. in cancer treatment). In recent years, there has been a focus to make use of marine organisms for this purpose. As a result, marine biotechnology is developing to meet the increasing demand. This thesis deals with one of the biomedically useful symbiotic soft corals, Sinularia flexibilis that co-exists with dinoflagellate algae or zooxanthellae, which together with the host (animal) form a holobiont. This species also produces compounds as intra-cellular photo-protective agents in shallow-water habitats; these compounds are called mycosporine-like amino acids (MAAs) with potential application in health care as sunscreens. An advantage of this coral is that from a parent colony of coral, a lot of clones may be obtained via asexual reproduction (fragmentation). If this procedure is used natural marine resources can be preserved because excessive sea harvest of the source coral is not necessary. Because there is an increasing demand for drugs such as antibiotics and anticancers of marine origin, the main goal of this research was to culture S. flexibilis at optimized captive conditions, so that they can supply, in part, the future demand for drug development. To have an overview of scientific research on the metabolites of this species, we reviewed studies into these secondary compounds and listed various alternatives for the captive cultivation of corals (Chapter 2). In this research, we studied the impact of environmental parameters such as irradiance (Chapter 3), the relevance of phototrophy and heterotrophy (Chapter 4), the effect of nutrient enrichment (Chapter 5) and of water velocity (Chapter 6) on the coral growth and physiology in captivity, and on the biosynthesis of secondary metabolites. As an alternative to the cultivation of whole organisms, the cultivation of coral cells is described in Chapter 7. - Secondary metabolites and cultivation opportunities (Chapter 2) To have an overview of scientific research on the secondary metabolites of this species, we reviewed studies into these compounds. Our review showed that a high percentage of these compounds are cytotoxic and may be promising as future anti-cancer drugs. To provide coral biomass, we discussed various approaches, including captive aquaculture. - Light-dependency (Chapter 3) As mentioned above, S. flexibilis is a symbiotic coral: it depends on the translocated photosynthates from its algal symbionts, the zooxanthellae. Accordingly, factors that promote photosynthesis of zooxanthellae should be given serious attention. Of these, light is being considered as the most important environmental factor affecting growth of symbiotic corals due to translocated photosynthates from zooxanthellae. Light intensity or photosynthetic photon flux density is important in promoting photosynthesis by zooxanthellae. Hence, finding optimum irradiance by applying various light intensities was the objective in the 3rd chapter of this thesis. Optimum specific growth rate (mu) of S. flexibilis was found at a range from 100 to 400 μmol quanta m⁻² s⁻¹. More or less constant mu over a range of light intensities was attributed to photoacclimation of the coral. The content of flexibilide, a major terpene of S. flexibilis, increased with light intensity; it showed lower contents at both very low and very high irradiances. The increase in flexibilide was explained as the coral’s response to stressful condition of high irradiances. The decrease in flexibilide content at the light extremes was ascribed to low photosynthesis due to weak and/or strong light intensities. - Phototrophy and heterotrophy (Chapter 4) The light-dependency of S. flexibilis and its zooxanthellae indicates that the algal photosynthates fuel metabolic requirements of the host coral. Our long-term findings verified this fact and showed that in the absence of light, even with addition of food , the coral was not able to grow nor to survive. Similarly, corals kept under optimal light without a continuous supply of ambient nutrient supply, were incapable to grow and survive. On the other hand, both colonies of S. flexibilis incubated at normal irradiances with or without extra feeding, grew well using available nutritional sources. In the same way, the coral physiology, i.e. the contents of zooxanthellae, chlorophyll, and MAAs reflected the effects of the above parameters. Therefore, both phototrophy and heterotrophy on ambient resources are important for S. flexibilis to perform optimally. In short-term, measurements of metabolic rate (photosynthesis and respiration) and estimating daily energy budget for S. flexibilis showed complete phototrophy. Estimating the daily ‘Contribution of Zooxanthellae to Animal Respiration (CZAR)’ yielded high values of zooxanthellae contribution to the host metabolism. Using energy equivalents for photosynthetically fixed carbon, we established that a small fraction of translocated fixed carbon is allocated to coral growth. Based on dependency of S. flexibilis on ambient nutritional sources in addition to irradiance, we concluded that photosynthates of zooxanthellae are deficient in growth-enhancing nutrients, especially nitrogenous compounds, which indeed need to be fulfilled heterotrophically for overall coral growth. - Nutrient enrichment and feeding (Chapter 5) To study the effect of essential nutrients enrichment (nitrogen and phosphorous) on the specific growth rate of S. flexibilis and its zooxanthellae, we performed prolonged experiments (Chapter 5). Our results showed that ammonium addition did not have any positive effect on coral growth; it reduced the growth of S. flexibilis at high levels. The coral was able to recover after high ammonium loadings. The effect of phosphate enrichment on the coral growth was neutral. Enrichment of this species with ammonium for three weeks resulted in an increase in both zooxanthellae and chlorophyll content after the 1st week of enrichment, followed by a decrease (equal to control) for the next two weeks. We concluded that zooxanthellae multiplication because of ammonium addition was temporary. - Flow-dependency (Chapter 6) The importance of water flow for sessile organisms such as S. flexibilis is to provide them with nutrition (e.g. suspended food particles), remove the coral’s wastes, and to facilitate exchange of substrates and products through modifying the boundary layer near their bodies. Long-term exposure of S. flexibilis to various water velocities resulted in the finding that the coral’s growth, physiology, and morphology are affected by flow regimes. Specific growth rates were optimal at an optimum water velocity of 11 cm s⁻¹. Based on the contents of zooxanthellae, chlorophyll, and total protein at the same water velocity for optimum growth, we concluded that optimal water velocity facilitated nutrient uptake through modification of the boundary layer thickness. This modification was aided by flexibility and swaying behaviour of S. flexibilis and also its polypary structure. High water velocities caused both coral and polyp retraction, which reduced exposure of its photosynthesizing surfaces together with decreased nutrient uptake resulting in low specific growth rates and also physiological parameters. - Coral cell culture (Chapter 7) We investigated cellular culture of S. flexibilis (Chapter 7) as an alternative to the coral supply, using different media and cell dissociation methodologies. A mechanical dissociation process provided the best method for the cell extraction procedure, maintaining always the highest number of cells extracted and subsequent cellular growth in all treatments. The best results from chemical reagents for dissociation was found using trypsin-EDTA. Coral cells obtained by spontaneous dissociation did not show signs of growth. Light was revealed as a fundamental parameter to be taken into account for the coral cell culture. The media GIM and GMIM displayed the best results for the maintenance of coral cell cultures. By means of a molecular test using Internal Transcribed Spacer (ITS) primers, the similarity of cultured coral cells and zooxanthellae at different culture media with those in the coral tissue was confirmed. In addition to possibility of the coral cell culture depending on both culture conditions and methodologies, our genetical cell identification test provided a reliable proof for the true cultured cells of S. flexibilis.