ADVANCES IN CULTURED SEAFOOD THROUGH EMBRYONIC STEM CELLS OF COMMERCIALLY RELEVANT MARINE SPECIES

Our overall goal is to develop embryonic cell lines from red drum (Sciaenops ocellatus) and Pacific whiteleg shrimp (Litopenaeus vannamei).Objective 1. Collection of fertilized embryos from red drum and Pacific whiteleg shrimp. Red drum broodstock will be spawned and embryos collected at various stages post-fertilization, with an emphasis on collecting embryos at the blastomere stage.Objective 2. Embryonic stem cell isolation. Embryonic stem cells will be isolated from fertilized eggs of each of the proposed species.Objective 3. Thraustochytrid control. Control of thraustochytrid spp. growth in marine invertebrate cultures is necessary; this objective will test a panel of antibiotics/antimycotics that are effective against thraustochytrid but do not have a negative impact on target cell growth.Objective 4. Biochemical characterization of egg contents. The biochemical composition of fertilized eggs will be analyzed with regard to fatty acid and amino acid profiles, total carbohydrate, as well as proximal analysis.Objective 5. Transcriptomic analyses. In this objective, we will conduct transcriptomic experiments to characterize cellular and genomic events occurring in cells at different stages in culture and in response to different media formulations.Objective 6. Spent media analysis. Nutrient utilization in cell culture samples will be analyzed at the same time points and media conditions as for transcriptomic analysis.Objective 7. Species targeted growth factors. In this objective, we will characterize growth factors based on sequence homology to identify homologous growth factors in the genome of each species.Objective 8. Morphological monitoring of cell growth over time. Cell growth properties, proliferation, and cell death will be monitored over time in cells cultured in different media formulations.Objective 9. Optimize media formulations. In this objective, we will develop medium formulations based on nutrient composition (Objective 4), growth factor analysis (Objective 7), spent media analysis (Objective 6), and results from monitoring cell growth over time (Objective 8).