ARELLANO, S.M.; TERWILLIGER, N.B.*; University of Oregon; University of Oregon: Hemocyanin, Cryptocyanin, and Phenoloxidase in Deep Sea (Bathynomous giganteus) and Intertidal (Cirolana harfordi) Isopods

The cirolanid isopods, Bathynomus giganteus and Cirolana harfordi, belong to the same family, but reside in very different habitats, the deep sea and the lower intertidal. Moreover, B. giganteus is purportedly an evolutionarily ancient isopod and thus may be a predecessor to C. harfordi. It has been suggested that structures of the closely related proteins, hemocyanin and cryptocyanin, could be used to distinguish phylogenetic relationships in the Ecdysozoa. Hemocyanin is a copper-containing, extracellular blood protein that combines reversibly with oxygen. Cryptocyanin is a copper-free hexameric protein similar in sequence and structure to hemocyanin that is involved in molting. This study compares the structure of these proteins in C. harfordi and B. giganteus. One- and two- hexameric hemocyanins are present in both isopods by non-denaturing pH 7.4 PAGE in the presence of Ca²⁺. This contrasts with published reports of an exclusively hexameric hemocyanin in B. giganteus. SDS-PAGE indicates at least two polypeptides with molecular masses between 73 and 83 kDa in each species, similar to hemocyanin subunits of other isopods. Western Blots identify a cryptocyanin-like protein in both isopods. These preliminary studies are the first to demonstrate the presence of cryptocyanin in the Peracarida. Hemocyanins of both isopods have phenoloxidase activity, and there is apparently no other phenoloxidase in the hemolymph, consistent with Pless et al. (ABB 409:402-410, 2003). This pattern is similar to chelicerate hemolymph but differs from that of other crustaceans, where both hemocyanin and a hemocyte phenoloxidase show enzymatic activity. Supported by NSF Grant IAB-9984202 (NBT) and NSF Grant OCE-0243688 (C.M. Young).