CROCKETT, E.L.*; HASSETT, R.P.; SHULTZ, R.W.; FUNK, K.R.; Ohio University, Athens: Preparation of Plasma Membranes from the Copepod Acartia hudsonica

Marine zooplankton, typically dominated by copepods (Class Crustacea), are a key link in the foodweb. Knowledge of how temperature and nutrition affect the biology of these animals is essential for understanding factors that limit growth. An animal’s sterol requirements are likely to be driven largely by contents of cholesterol in plasma membranes. In our ongoing study of sterol limitation of zooplankton growth we are investigating the nature of cholesterol distribution in the plasma membrane of the copepod. This requires an enriched preparation of plasma membranes from the animals. Plankton were collected from Frenchman Bay, Maine (near the Mount Desert Island Biological Laboratory) and Acartia hudsonica were sorted by relying, in part, on the positive phototaxis of these copepods. One to two grams of starting material (a minimum of 10,000 animals) were used for each membrane preparation. Animals were homogenized and homogenates were subjected to differential and density-gradient centrifugation in order to obtain a fraction that contained partially purified plasma membranes. Analyses of marker enzymes for plasma and intracellular membranes were used to determine the degree of plasma membrane enrichment. In the final membrane fraction, enrichment factors of the marker for plasma membranes Na⁺/K⁺-ATPase were 6-fold (0.4 SEM; n=6) while mitochondrial and ER markers (succinate-cytochrome c reductase and NADPH-cytochrome c reductase, respectively) were enriched only 2-fold. These results indicate a significant and consistent enrichment of plasma membranes from the copepod. Supported by NSF OCE 0117132.