Yasuyuki Arakane, Biochemistry

This proposal describes a comprehensive functional genomics study of the insect dopachromeconversion enzyme (DCE, yellow) gene family and its role in cuticle pigmentation and sclerotization using the red flour beetle, Tribolium castaneum, as the model insect species. This important gene family has been studied in Drosophila but hardly at all in any other species.

DCE is involved in the melanin biosynthetic pathway and significantly accelerates pigmentation reactions. In Drosophila melanogaster (Dm), 13 genes have been annotated as members of the yellow gene family and recent studies suggest that Dmyellow-y is involved in melanin production. However, the physiological functions of the rest of the yellow genes are unknown. From an exhaustive search of the Tribolium genome, I have identified 14 genes encoding yellow-like proteins including the Dmyellow-y ortholog. The functions and roles of each Tribolium yellow gene in cuticle pigmentation and sclerotization as well as insect growth and development will be analyzed by the following experimental strategy:
1) Clone full-length cDNAs corresponding to all fourteen of the yellow genes.
2) Determine the expression patterns of the yellow genes during development (from embryonic through adult stages) and also their tissue-specificities of expression by realtime PCR (qPCR) and by tiling array analyses.
3) Down-regulate transcripts for individual yellow genes by injection of gene-specific, double-stranded RNAs (dsRNAs) and observe any resulting phenotypic changes.
4) Analyze the mechanical properties of abnormal elytra resulting from the RNAi experiments using a TA Dynamic Mechanical Analyzer (DMA).

These experiments will provide critical information on cuticle formation and maturation and could also open up new strategies for insect control, which target cuticle biochemistry.

Data obtained from this grant will be used to support a full proposal to agencies such as NSF or USDA to investigate the regulation of expression of insect yellow genes and to pursue a comprehensive understanding of their roles in cuticle pigmentation and stabilization.