Panning for sperm gold: Isolation and purification of apyrene and eupyrene sperm from lepidopterans

We describe a simple and straightforward procedure for the purification and separation of apyrene and eupyrene forms of lepidopteran sperm. The procedure is generally applicable to both butterfly and moth species with results varying according to the relative amounts of sperm produced and size of sperm storage organs. The technique relies upon inherent differences between eupyene sperm bundles and free apyrene sperm morphology. These differences allow for separation of the sperm morphs by repeated “panning” of sperm bundles into the center of a plastic dish. The purified eupyrene sperm bundles can then be removed and apyrene sperm collected from the supernatant by centrifugation. Efficacy of the purification process was confirmed by light microscopy and gel electrophoresis of the resulting fractions. Both one- and two-dimensional gel electrophoresis identified significant protein differences between the fractions further suggesting that the panning procedure effectively separated eurpyrene from apyrene sperm. The panning procedure should provide a convenient and accessible technique for further studies of sperm biology in lepidopterans.