Microalgae diets for landbased aquaculture of the cockle Cerastoderma edule: impacts of dietary fatty acids on growth

Land-based shellfish culture as a part of a multi-trophic aquaculture systems has yet to be implemented in Europe. Recently the pilot project Zeeuwse Tong (The Netherlands) evaluated the feasibility of a system of fish (Dover sole), ragworms, phytoplankton and bivalves. This thesis focused on the dietary fatty acids impact on growth, survival and fatty acid composition of juveniles (shell length >5mm) of the common cockle <em>Cerastoderma edule</em>, for land-based culture. Dietary fatty acids were chosen as the main nutritional research subject given the existing literature indicating their importance for reproduction, and growth and survival of larvae, post-larvae, spat and seed of most bivalves. However, since grow-out of juvenile cockles in land-based aquaculture is not common, no information is available on the dietary fatty acid requirements of juveniles. To determine if the presence of specific fatty acids is more important for growth and survival of juvenile cockles than the total amount of dietary fatty acids supplied, least cost linear programming was used to design live microalgae diets. Monocultures of indigenous algae were grown under controlled conditions and sampled to determine their quality in terms of dry weight, organic matter, lipid and fatty acids composition. These differences in biochemical composition of the microalgae were used in a least-cost programming software, and allowed the formulation of three diets with different fatty acid contents, but similar dry weight, organic matter, lipid and total fatty acid content. The formulated diets were then used to determine the importance of dietary fatty acids for growth, survival and fatty acid composition of juvenile cockles (<em>Cerastoderma edule</em>). Juvenile cockles (6.24±0.04 mm) were constantly fed live microalgal diets with similar lipid, organic matter and total fatty acid contents but different individual fatty acid concentrations: high-EPA, no-DHA and low-ARA diets. Growth was positively affected by high-EPA and low-ARA diets, whereas no significant growth was observed for the no-DHA diet. However, high mortality of cockles fed no-DHA diet and the low absorption efficiency and clearance rate of this diet indicate that this diet is unsuitable for juvenile cockles. In balanced diets with EPA and DHA, lower concentrations of ARA did not limit growth. The combination of EPA and DHA in a live microalgae diet was beneficial for the growth and survival of juvenile cockles, but no conclusion on the importance of supplying only EPA could be made. To address this, an additional growth experiment was designed. Growth and fatty acid profiles of juveniles cockles were determined after 28 days of feeding diets:1) with EPA 2) with DHA; 3) with EPA and DHA from a mixture of a diatom with a green algae; 4) with EPA and DHA from a mixture of the two green algae and 5) without long-chain (>C20) fatty acids. Significantly higher growth rate was observed with the treatment rich in EPA, while the lowest growth rates were found in without EPA. These results indicate that EPA is necessary for the short-term growth of cockles. Moreover, the fatty acid profile of the polar lipids confirmed that, like most marine invertebrates, juvenile <em>C. edule </em>are not able to biosynthesize EPA and DHA from 18:3n-3 but are capable of biosynthesizing diene and triene NMI fatty acids. The occurrence of the NMI fatty acids was diet related, thus refuting the possibility of production of NMI as replacement for EPA and DHA. Furthermore, in order to develop land-based culture of cockles it is crucial to have a supply of high quality live microalgal diets produced with minimum effort. Therefore, the use of simplified microalgae media, with different nitrogen sources (nitrate or ammonium) and molar N:P ratio, phosphorus, silica, iron, manganese and vitamins were also investigated. <em>Chaetoceros muelleri</em>, a marine diatom that was chosen given its robustness and quality as food for juvenile cockles, reacted positively to all simplified media. At the exponential phase, all cultures had reached similar cellular concentrations and dry weight productions. Cultures grown on ammonium media had a longer cultivation period, 20 days. Considering dry-weight production, culture duration, nutrient efficiency and lipid composition, the simplified media containing ammonium, phosphorus, silica, iron, manganese and vitamins proved to be a viable choice for batch culture of <em>C. muelleri.</em> The choice between these two media depends on the final purpose of the microalgae cultures and whether lipid contents (NH49:1), dry weight biomass (NH425:1) or nitrogen input and output (NH49:1) are more important. In this thesis it was demonstrated that mono-algal diets with high EPA, as well as mixed live microalgae diets with high EPA and DHA contents proved to be suitable diets for <em>C. edule </em>juveniles. In addition, a positive relation between growth and the amount fed was found. The growth rates of the cultured cockles in this thesis are in accordance with the rates observed under natural conditions. The use of the simplified microalgae culture medium tested represents a significant decrease in the cost and preparation of the medium, as well as longer production and harvest periods, while maintaining microalgae quality. These results indicate that land-based aquaculture of cockles and microalgae is therefore a promising venture, with potential for expansion.