René H. Wijffels

Cultivation of Marine Sponges

Abstract: There is increasing interest in biotechnological production of marine sponge biomass owing to the discovery of many commercially important secondary metabolites in this group of animals. In this article, different approaches to producing sponge biomass are reviewed, and several factors that possibly influence culture success are evaluated. In situ sponge aquacultures, based on old methods for producing commercial bath sponges, are still the easiest and least expensive way to obtain sponge biomass in bulk. However, success of cultivation with this method strongly depends on the unpredictable and often suboptimal natural environment. Hence, a better-defined production system would be desirable. Some progress has been made with culturing sponges in semicontrolled systems, but these still use unfiltered natural seawater. Cultivation of sponges under completely controlled conditions has remained a problem. When designing an in vitro cultivation method, it is important to determine both qualitatively and quantitatively the nutritional demands of the species that is to be cultured. An adequate supply of food seems to be the key to successful sponge culture. Recently, some progress has been made with sponge cell cultures. The advantage of cell cultures is that they are completely controlled and can easily be manipulated for optimal production of the target metabolites. However, this technique is still in its infancy: a continuous cell line has yet to be established. Axenic cultures of sponge aggregates (primmorphs) may provide an alternative to cell culture. Some sponge metabolites are, in fact, produced by endosymbiotic bacteria or algae that live in the sponge tissue. Only a few of these endosymbionts have been cultivated so far. The biotechnology for the production of sponge metabolites needs further development. Research efforts should be continued to enable commercial exploitation of this valuable natural resource in the near future.

The Biology and Economics of Coral Growth

To protect natural coral reefs, it is of utmost importance to understand how the growth of the main reef-building organisms—the zooxanthellate scleractinian corals—is controlled. Understanding coral growth is also relevant for coral aquaculture, which is a rapidly developing business. This review paper provides a comprehensive overview of factors that can influence the growth of zooxanthellate scleractinian corals, with particular emphasis on interactions between these factors. Furthermore, the kinetic principles underlying coral growth are discussed. The reviewed information is put into an economic perspective by making an estimation of the costs of coral aquaculture.

Towards Commercial Production of Sponge Medicines

Sponges can provide potential drugs against many major world-wide occurring diseases. Despite the high potential of sponge derived drugs no sustainable production method has been developed. Thus far it is not fully understood why, when, where and how these metabolites are produced in sponges. For the near future sea-based sponge culture seems to be the best production method. However, for controlled production in a defined system it is better to develop in vitro production methods, like in vitro sponge culture or even better sponge cell culture, culture methods for symbionts or the transfer of production routes into another host. We still have insufficient information about the background of metabolite production in sponges. Before production methods are developed we should first focus on factors that can induce metabolite production. This could be done in the natural habitat by studying the relation between stress factors (such as predation) and the production of bioactive metabolites. The location of production within the sponge should be identified in order to choose between sponge cell culture and symbiont culture. Alternatively the biosynthetic pathways could be introduced into hosts that can be cultured. For this the biosynthetic pathway of metabolite production should be unraveled, as well as the genes involved. This review discusses the current state of sponge metabolite production and the steps that need to be taken to develop commercial production techniques. The different possible production techniques are also discussed.

Primmorphs from seven marine sponges: formation and structure

Primmorphs were obtained from seven different marine sponges: Stylissa massa, Suberites domuncula, Pseudosuberites aff. andrewsi, Geodia cydonium, Axinella polypoides, Halichondria panicea and Haliclona oculata. The formation process and the ultra structure of primmorphs were studied. A positive correlation was found between the initial sponge-cell concentration and the size of the primmorphs. By scanning electron microscopy (SEM) it was observed that the primmorphs are very densely packed sphere-shaped aggregates with a continuous pinacoderm (skin cell layer) covered by a smooth, cuticle-like structure. In the presence of amphotericin, or a cocktail of antibiotics (kanamycin, gentamycin, tylosin and tetracyclin), no primmorphs were formed, while gentamycin or a mixture of penicillin and streptomycin did not influence the formation of primmorphs. The addition of penicillin and streptomycin was, in most cases, sufficient to prevent bacterial contamination, while fungal growth was unaffected.

Cultivation of sponges, sponge cells and symbionts: achievements and future prospects.

Marine sponges are a rich source of bioactive compounds with pharmaceutical potential. Since biological production is one option to supply materials for early drug development, the main challenge is to establish generic techniques for small-scale production of marine organisms. We analysed the state of the art for cultivation of whole sponges, sponge cells and sponge symbionts. To date, cultivation of whole sponges has been most successful in situ; however, optimal conditions are species specific. The establishment of sponge cell lines has been limited by the inability to obtain an axenic inoculum as well as the lack of knowledge on nutritional requirements in vitro. Approaches to overcome these bottlenecks, including transformation of sponge cells and using media based on yolk, are elaborated. Although a number of bioactive metabolite-producing microorganisms have been isolated from sponges, and it has been suggested that the source of most sponge-derived bioactive compounds is microbial symbionts, cultivation of sponge-specific microorganisms has had limited success. The current genomics revolution provides novel approaches to cultivate these microorganisms.

Adaptation of a Madin–Darby canine kidney cell line to suspension growth in serum-free media and comparison of its ability to produce avian influenza virus to Vero and BHK21 cell lines

Madin-Darby canine kidney (MDCK) cells are currently considered for influenza vaccine manufacturing. A drawback of these cells is their anchorage dependent growth, which greatly complicates process scale-up. In this paper a novel MDCK cell line (MDCK-SFS) is described that grows efficiently in suspension and retained high expression levels of both α-2,6 and α-2,3 sialic acid receptors, which bind preferably to human and avian influenza viruses, respectively. The production of avian influenza virus by BHK21, Vero and MDCK-SFS cell lines was compared. Although BHK21 cells consisted of two populations, one of which lacks the α-2,3 receptor, they supported the replication of two influenza strains to high titres. However, BHK21 cells are generally not applicable for influenza production since they supported the replication of six further strains poorly. MDCK-SFS cells yielded the highest infectious virus titres and virus genome equivalent concentration for five of the eight influenza strains analyzed and the highest hemagglutination activity for all eight virus strains. Taken together with their suitability for suspension growth this makes the MDCK-SFS cell line potentially useful for large scale influenza virus production.

Hypothesized kinetic models for describing the growth of globular and encrusting demosponges

The marine sponges Dysidea avara and Chondrosia reniformis (globular forms) were cultured in the laboratory on a diet of viable Phaeodactylum tricornutum cells and dissolved nutrients (algae and fish powders). Our growth data were combined with literature data for Pseudosuberites andrewsi (a globular sponge) and for the encrusting sponges Oscarella lobularis, Hemimycale columella, and Crambe crambe. The suitability of three growth models—linear, exponential, and radial accretive—for describing the growth of globular and encrusting sponges was assessed. Radial accretive growth was determined to be the best model to describe growth of both encrusting and globular sponges. Average growth rates of 0.051 ± 0.016 and 0.019 ± 0.003xa0mm/day (calculated as the increase of the radius of the sponge per day) were obtained experimentally for D. avara and C. reniformis, respectively.

Sponge-cell culture? A molecular identification method for sponge cells.

AbstractDissociated sponge cells are easily confused with unicellular organisms. This has been an obstacle innthe development of sponge-cell lines. We developed a molecular detection method to identify cells of thensponge Dysidea avara in dissociated cell cultures. The 18S ribosomal RNA gene from a Dysidea avara specimennwas sequenced and compared to eukaryotic 18S rDNA sequences picked up from a proliferating cell culturenthat originated from a dissociated Dysidea avara specimen. Our method proved unambiguously that this wasnnot a sponge-cell culture. Therefore, it provides a valuable tool for further research on sponge-cell cultures.

Cell cycle analysis of primary sponge cell cultures

Proliferation of sponge cells is generally measured via cell counts or viability assays. However, more insight into the proliferative state of a sponge cell population can be obtained from the distribution of the cells over the different phases of the cell cycle. Cell cycle distribution of sponge cells was measured via flow cytometry after staining the DNA with propidium iodide. The five sponges studied in this paper all showed a large fraction of cells in G1/G0 compared to G2/M and S, indicating that cells were not actively dividing. In addition, some sponges also showed a large apoptotic fraction, indicating cell death. Additional apoptosis measurements, based on caspase activity, showed that harvesting and dissociation of sponge tissue to initiate a primary cell culture was directly correlated with an increase in apoptotic cells. This indicates that for the development of cell cultures, more attention should be given to harvesting, dissociation, and quality of starting material. Finally, cultivation conditions used were ineffective for proliferation, since after 2xa0d of cultivating Haliclona oculata cells, most cells shifted towards the apoptotic fraction, indicating that cells were dying. For development of in vitro sponge cell cultures, flow cytometric cell cycle analysis is a useful method to assess the proliferative state of a sponge cell culture and can be used to validate improvements in harvesting and dissociation, to select sponges with good proliferative capacities and to study the influence of culture conditions for stimulating cell growth.