Crustacean hemocyanin gene family and microarray studies of expression change during eco-physiological stress

Proteins in the arthropod hemocyanin gene family are involved in major physiological processes, including aerobic respiration, the innate immune response, and molting. Members of this family, hemocyanin, cryptocyanin, and phenoloxidase, are multisubunit molecules that assemble into hexamers and higher aggregates. The hemocyanin hexamers show species-specific subunit heterogeneity. It is hypothesized that this subunit diversity is maintained as a mechanism of selection for functional diversity under changing developmental and environmental conditions. There is good evidence for a strong relationship between subunit composition and functional diversity in the hemocyanins. We have amplified, cloned, and sequenced the complete cDNAs of the 6 hemocyanin genes, 2 cryptocyanins, and 1 phenoloxidase of Cancer magister. Alignment of the amino acid sequences provides the first opportunity to assess in 1 species of brachyuran crustacean the similarities and differences among all the hemocyanin subunits and compare them with cryptocyanin and phenoloxidase. A phylogeny of sequences of crustacean members of the arthropod hemocyanin gene family is described. Construction of a cDNA library for C. magister microarray studies is in progress. The microarrays will be queried using transcriptional profiles from crabs sampled during developmental, molting, and physiological perturbations. The combination of genomics, proteomics, and gene-by-gene analyses will help us dissect how much a gene sequence in this hemocyanin family can vary while conserving function and which aspects of preservation of shape and structural flexibility are essential for functional stability. Integrating focused gene studies with global-expression profiling can eventually lead to the identification of functional networks at the level of the gene, the multisubunit molecule, and the whole organism.