VIERTHALER, L; RYAN, M; TERWILLIGER, N.B.*; Southwestern College, Winfield, Kansas; University of Oregon, Oregon Institute of Marine Biology, Charleston; University of Oregon, Oregon Institute of Marine Biology, Charleston: A functional hemocyanin in the leptostracan Nebalia: Insights into molecular and arthropod phylogenies.

In the Crustacea, the subclass Malacostraca consists of both the Eumalacostraca, which includes large groups such as the decapods, amphipods and isopods, and the Phyllocarida, which includes the order Leptostraca, a very small group often considered the most basal of the existing malacostracans. Hemocyanin, the copper based oxygen binding protein, is widely distributed among the Arthropoda, but in the Crustacea, it has been described only in the eumalacostrachans. We wondered whether the leptostracan Nebalia sp, a small pinkish crustacean found in hypoxic environments under boulders in the low intertidal zone along the Oregon coast, would express a hemocyanin (Hc) similar to the more advanced malacostracans. Alternatively, it might express hemoglobin as do the Branchiopoda, another crustacean group to which the leptostracan Phyllocarida have sometimes been assigned. We found that Nebalia has a Hc composed of 3 chains whose subunit masses are similar to other crustacean Hcs according to SDS PAGE. The molecule, purified by DEAE BioGel, is a hexamer as determined by pH 7.4 PAGE and by TEM. The Hc reacts with an anti-Hc/anti-cryptocyanin Ab on Western blots, and the cDNA sequence firmly establishes that it is a Hc with conserved copper binding sites. Most importantly, we have shown that the molecule reversibly binds oxygen. This functional hemocyanin in a leptostracan is the most ancestral crustacean Hc described and confirms the phylogeny of the phyllocaridans in the Malacostraca. Supported by NSF 9984202 to NBT.