Hemolymph analysis and evaluation of newly formulated media for culture ofshrimp cells (Penaeus stylirostris)

Creation of a shrimp cell line has been an elusive goal. This failure may b e due to the composition of the cell culture medium, which may be inadequat e to support primary cultured cells. Shrimp hemolymph should contain the nu tritional components needed to support cell growth and division. We report here the comprehensive biochemical analysis of hemolymph from the blue shri mp, Penaeus stylirostris (Litopenaeus stylirostris) (see Holthuis, L. B. Sh rimps and prawns of the world, in: FAO species catalog. Vol. 1. Rome: Food and Agriculture Organization of the United Nations; 1980), for free amino a cids (FAAs), carbohydrates, electrolytes, metals, pH, and osmolality. Level s of hemolymph components were compared to 2XL-15 with 20% fetal bovine ser um, a commonly used culture medium for crustacean cells. The FAAs, taurine and proline, and the metals, strontium and zinc, were significantly higher in hemolymph than in the 2XL-15 medium. In contrast, other FAAs were up to 50 times higher in the 2XL-15 medium than in the hemolymph. To mimic more c losely the hemolymph composition, we created two new media based on either the 0.2XL-15 or the M199 medium. We compared the microscopic appearance of cells cultured in these media and evaluated deoxyribonucleic acid (DNA) and protein synthesis by H-3-thymidine uptake and S-35-methionine uptake assay s. The ovary cells of P. stylirostris cultured in either of the new media f ormed monolayers, while the cells cultured in 2XL-15 medium did not. Despit e these differences, there was no evidence of sustained DNA or protein synt hesis with any of the media. Future studies to establish a shrimp cell line should focus on analysis of the cell cycle and on overcoming the molecular blocks to cell division.