Identification, mRNA expression and functional analysis of several yellow family genes in Tribolium castaneum

Querying the genome of the red flour beetle, Tribolium castaneum, with the Drosophila melanogaster Yellow-y (DmY-y) protein sequence identified 14 Yellow homologs. One of these is an ortholog of DmY-y, which is required for cuticle pigmentation (melanization), and another is an ortholog of DmY-f/f2, which functions as a dopachrome conversion enzyme (DCE). Phylogenetic analysis identified putative T. castaneum orthologs for eight of the D. melanogaster yellow genes, including DmY-b, -c, -e, -f, -g, -g2, -h and -y. However, one clade of five beetle genes, TcY-1-5, has no orthologs in D. melanogaster. Expression profiles of all T. castaneum yellow genes were determined by RT-PCR of pharate pupal to young adult stages. TcY-b and TcY-c were expressed throughout all developmental stages analyzed, whereas each of the remaining yellow genes had a unique expression pattern, suggestive of distinct physiological functions. TcY-b, -c and -e were all identified by mass spectrometry of elytral proteins from young adults. Eight of the 14 genes showed differential expression between elytra and hindwings during the last three days of the pupal stage when the adult cuticle is synthesized. Double-stranded RNA (dsRNA)-mediated transcript knockdown revealed that TcY-y is required for melanin production in the hindwings, particularly in the region of the pterostigma, while TcY-f appears to be required for adult cuticle sclerotization but not pigmentation.