Silk production has independently evolved in numerous arthropod lineages, such as Lepidoptera, the moths and butterflies. Lepidopteran larvae (caterpillars) synthesize silk proteins in modified salivary glands and spin silk fibers into protective tunnels, escape lines, and pupation cocoons. Molecular sequence data for these proteins are necessary to determine critical features of their function and evolution. To this end, we constructed an expression library from the silk glands of the ghost moth, Hepialus californicus, and characterized light chain
fibroin and heavy chain fibroin gene transcripts. The predicted H. californicus silk fibroins share many elements with other lepidopteran and trichopteran fibroins, such as conserved placements of cysteine, aromatic, and polar amino acid residues. Further comparative analyses were performed to determine site-specific signatures of selection and to assess whether fibroin genes are informative as phylogenetic markers. We found that purifying selection has constrained mutation within the fibroins and that light chain fibroin is a promising molecular marker. Thus, by characterizing the H. californicus fibroins, we identified key functional amino acids and gained insight into the evolutionary processes that have shaped these adaptive molecules.