The seagrass Posidonia oceanica (L.) Delile forms widespread meadows, which exert a crucial role in the Mediterranean coastal waters. The process of P. oceanica transplantations is complex and a clear and consistent planning and implementation approach was lacking. The guidelines proposed here described step by step an efficient procedure for the meadows rehabilitation. - The selection of the site will influence the results of the transplantation (mortality, shoot loss, rooting speed). A similarity of environmental conditions of donor and recipient beds must exist (temperature, salinity) with respect to the ecological requirements of P. oceanica. The sites highly impacted by seawage discharges (in the vicinity of fish farming) are unsuitable for seagrass restoration plan, until the installation of efficient waste water treatment. It is essential to take into account the substrate-energy regime (i.e. wave, current) of the site. Avoid rocky and muddy sediment. Avoid the sites with high hydrodynamic pressure. Substrates with mean granulometry higher than 1cm are generally zones where hydrodynamic pressures are too high to permit the transplantation. When transplantation occurs on high hydrodynamic pressures zones, grids are dig up, the shoots have difficulties to take root, and the leaves are damaged. The shoot mortality is thus higher than on low hydrodynamic pressure sites. Ideal conditions are small sandy areas (50 to 100m²) in the natural P. oceanica meadows. - The seeds are often rare in some area and seedling show low survival rate. P. oceanica cuttings from deeper zones, adapted to low light and transplanted at higher light intensity in shallow water are more resistant. The use of natural uprooted shoots during storms or after a breaking down of cliff, limits the impact of transplant collection on the healthy meadow. The gathering of shoots in the vicinity of the transplanted area will ensure better results. - Choose pieces of horizontal (plagiotropic) rhizomes (10cm long) bearing maximum 3 shoots. The most successful transplants is obtained with a 10 -15cm rhizomes length. On longer rhizome, the mortality of shoots is more frequent and the rhizome has a tendency to deterioration probably induced by bacterial growth spreading and infection of meristematic zones. Cut the long roots, they are often broken during the installation of the rhizomes on the grids and they tend to get rotten during the first month after the transplantation, (cuttings with root buds are preferable). - To enhance the stability of the sediment and to fix the shoots, the rhizomes have to be attached on grids. The use of unanchored shoots planted by hand into the sediment, like for Z. marina is not suitable for P. oceanica. The grids (mesh size: 10x10 or 5x5cm) of one square meter must be underwater fixed on the sediment. - The detailed electrification procedure (material installation, technical instructions) is described in the section XX in this volume. If the shoot installation precedes the electrification and if metallic links are used, the link must be placed as far as possible from the meristematic zones. A deterioration of this zone has been noted during electrification process. - In calm areas, it is possible to use organic link (Sisal rope), the rooting of shoots go faster than the disintegration of the organic link. However, in high hydrodynamic pressure areas, the rooting is more difficult (and slower), so the use of metallic or plastic links which will attach the rhizome until a solid rooting (two years) is preferable. The link ensures the stability of the unrooted rhizome on the transplantation grid during the storms, it also protect the shoots against the effect of the fauna (grazers of sediment and burrowers). - A density of twenty to thirty pieces of rhizomes/m² (each with one to three shoots) provides a good barrier effect by the leaves and promotes the rooting. In choppy areas, the increasing of transplanted rhizome (fifty) enhances the barrier effect of leaves against the water currents and decreases any sediment deficit. - The alignment of rhizomes must be avoided to reduce the possibility of erosion. The rhizomes have to be attached paralleled and diagonally along the grid axis. - The transplantation shows good results during all the year. But before and after the storm periods, the marine gardeners must verify the fixation of the shoots on the grids. The multiplication of shoots, the ramification of rhizomes occurs essentially between March and September. When temperature exceeds 20°C, the shoot mortality is high. - Time used: -- Collect of cuttings 1h/4m² underwater work. -- Selection of cuttings 1h/4m² Lab works. -- Installation of grids in situ 1h/4m² underwater work. -- Installation of cuttings on the grids 2h/4m² underwater work. -- Survey of grids 1h/4m underwater work.
The technologies for the farming of marine sponges developed in the course of the project build the second product branch. There are two different technologies available. On one hand the already patented metal-net technology where the sponge cuttings are held between the meshes of a fishery net. In addition several tools were developed to adapt the environmental conditions species specific. On the other hand the ERCON (Electrochemical Reef Construction) technology was used to build constructions for the farming of substrate depended and encrusting sponge species e.g. the collagen source Chondrosia reniformis. The field of application is basically built by companies and institutes that are working in the research and in the production of marine natural products. Their interest in these technologies is supposed to be strongly depended on their interest in sponge natural products. Whereby sponges are only one possible marine organism that could be farmed by these technologies. Corals, tunicates and algae might be additional target organisms. To our knowledge no other patented and working mariculture technologies are available on the market so far. The general practice is that the quest for novel compounds from the sea did not begin until analytical techniques had been developed requiring much smaller quantities of marine life - one to three kilograms - that can be grown sustainable in aquaculture. In addition there is still a strong interest from industries in aquaculture techniques to overcome the supply problem. The application potential of the ERCON technology could be seen as well in projects that concentrate on the upvaluation of coastal sand areas with artificial reef structures.
We have investigated several sponges collected in the bay of Calvi and in Rovinj, which were all subjected to the above procedure. We have focused our interest on the natural constituents of the species reported below, whose chemical investigation allowed to isolate a number of bioactive secondary metabolites, some of them being new compounds. All the isolated metabolites were fully structurally characterized through an extensive application of different chemical and spectroscopic analytical methods, including mass spectrometry and largely mono and bidimensional Nuclear Magnetic Resonance (NMR) techniques (1H-NMR, 13C-NMR, 1H-1H COSY, HOHAHA, ROESY, HSQC, HMBC). These modern two-dimensional techniques improved NMR spectroscopy as a vital tool for the marine natural product chemist since provide decisive information for the elucidation of complex structures, including the stereochemical details, on just a few milligrams of a reasonably pure product.
Collagen was isolated from different marine sponges and characterized. In addition, sponge enzymes involved in formation of the sponge skeleton including sponge collagen have been cloned. Collagen was also isolated from farmed sponge specimen. The content was between 15% (Ircinia variabilis) and 23% (Agelas oroides; lyophilized collagen sediment in relation to lyophilised sponge material). Sponge collagen has several advantages compared to collagen from other (mainly bovine) sources. A protease, which is most likely involved in collagen catabolism in sponges, was purified and characterized.