Comparison Of Hemolymph Proteins From The Horseshoe Crab, Limulus polyphemus     Ruth Barratt and Stephen A. Smith   Aquatic Medicine Laboratory, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0442 USA     The horseshoe crab (Limulus polyphemus) is found along the Atlantic coast of the U.S. from Maine to the Gulf of Mexico. The horseshoe crab has been described as one of the oldest living organisms on the planet, with it’s closest relatives, based on morphological characteristics, being in the Subphylum Chelicerata, which contains the scorpions and spiders. To further examine this relationship, hemolymph from several adult horseshoe crabs was analyzed by SDS-PAGE and the protein bands separated. The bands were then compared to protein bands derived from the hemolymph of an emperor scorpion, spider, lobster and blue crab. Results of the SDS-PAGE analysis of horseshoe crab hemolymph produced approximately three major proteins that most likely represent the alpha-2 macroglobulin, hemocyanin and C-reactive protein, respectively. Western blot analysis was performed using a rabbit anti-horseshoe crab whole hemolymph antibody and the results demonstrated that the rabbit antibody clearly recognized all of the horseshoe crab protein bands in the gel. In addition, the rabbit anti-horseshoe crab antibody also recognized protein bands from the hemolymph of the emperor scorpion and spider, but not those of the lobster and blue crab. This is further evidence that members of the Class Arachnida are more closely related to the horseshoe crab than are members of the Subphylum Crustacea.