The purpose of this grant is to study the developmental changes that occur in structure and function of hemocyanin, the blue, copper containing respiratory protein that is found dissolved in the blood of arthropods and molluscs. Respiratory proteins, hemocyanins, hemoglobins and hemerythrins, are excellent models for unique insights into the structure of oxygen transport molecules and how the molecules combine reversibly with oxygen. A dramatic change in hemocyanin subunit composition and oxygen binding properties occurs during development from late larval stage, or megalopa, to adult in the locally abundant Dungeness crab, Cancer magister. Hemocyanin expression in the unfertilized egg or oocyte, the fertilized embryo, and the newly hatched larval stage or zoea, will be analyzed to determine when changes in hemocyanin expression occur. Where and how the hemocyanin molecules are synthesized will be determined in the crab. Larval Dungeness crabs are active swimmers in the well oxygenated surface waters of estuaries and open ocean whereas the adult crab is found buried or walking along the less oxygenated muddy sandy bottom of estuaries and the nearshore. Accordingly, experiments in both laboratory and field will ask how a stage specific hemocyanin might be of help to the crab at a particular time in its life cycle and how the protein enables the animal to respond to environmental stresses such as temperature, salinity and low oxygen. Metabolic studies will also be done to see whether the functional properties of larval hemocyanin are related to the physiological demands of the larva versus the adult. These studies will provide models to explore the relationship between a protein's structure and its functional properties, a central question in understanding how protein molecules work. Information on the site and manner of hemocyanin synthesis will help to increase our knowledge of other medically important copper containing proteins such as cytochrome oxidase. The relationship of respiratory proteins to one another and to other families of proteins is of great interest to the field of evolutionary biology. In addition, the Dungeness crab is a commercially important fishery in the Pacific Northwest. Information about the crab's development and the role hemocyanin plays in its tolerance to various environmental conditions will enhance management of the natural fishery as well as efforts to develop a viable Dungeness crab aquaculture industry.
