Complete sequence, expression, and evolution of the hexamerin LSP-2 of Calliphora vicina

In cyclorraphan Diptera, two different types of hemolymph proteins exist which belong to the hexamerin family. During the last larval instar, Calliphora vicina synthesizes, besides the major fraction of arylphorin, a second hexameric protein, LSP-2. Here the developmentally regulated biosynthesis of this protein was analyzed. Western blot analyses showed that LSP-2 is not present in eggs, 1st, and 2nd instar larvae, whereas it can be detected in all tissues of last instar larvae. We report the characterization of the complete cDNA sequence that encodes a LSP-2 subunit, a nascent polypeptide of 701 amino acids with a molecular mass of 83.16 kDa. By Northern blotting, a mRNA of about 2.2 kb coding for LSP-2 is identified exclusively in the fat body of 3rd larval instars reflecting the stage and tissue specificity of LSP-2 gene expression. Phylogenetic analysis demonstrates the existence of two distinct groups of hexamerins in Diptera.