Molecular characterization of hemocyanin and hexamerin from the firebrat Thermobia domestica (Zygentoma).

Hexapods possess a tracheal system that enables the transport of oxygen to the inner organs. Although respiratory proteins have been considered unnecessary in most Hexapoda for this reason, we recently showed the presence of a functional hemocyanin in the stonefly Perla marginata. Here we report the identification and molecular characterization of a hemocyanin from Zygentoma (Thysanura). We obtained the full length cDNA of two distinct subunit types from the firebrat Thermobia domestica, and partial sequences of the orthologs from the silverfish Lepisma saccharina. The native T. domestica hemocyanin subunits both consist of 658 amino acids, but a signal peptide for transmembrane transport is missing in subunit 2. In adult firebrats both hemocyanin subunits represent a substantial proportion of the total hemolymph proteins. Phylogenetic analyses show that the subunit types are orthologous to subunits 1 and 2 of the stonefly Perla marginata. We further identified and sequenced a hexamerin subunit from T. domestica (689 amino acids), which suggests an early emergence of this type of proteins in hexapod evolution. In contrast to most other hexamerins, it does not reveal a high content in phenylalanine and tyrosine, which may be interpreted that the accumulation of aromatic amino acids commenced later in hexamerin evolution. Molecular clock calculations using hexamerins suggest that the divergence of Zygentoma and Pterygota occurred around 387 million years ago, which is in excellent agreement with the available fossil record.