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Title: LACCASE 2 IS THE PHENOLOXIDASE GENE REQUIRED FOR BEETLE CUTICLE TANNING

Author
item ARAKANE, YASUYUKI - KANSAS STATE UNIV
item MUTHUKRISHNAN, SUBBARATNAM - KANSAS STATE UNIV
item Beeman, Richard
item KANOST, MICHAEL - KANSAS STATE UNIV
item KRAMER, KARL - 5430-05-30 RETIRED

Submitted to: Proceedings of the National Academy of Sciences (PNAS)
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/31/2005
Publication Date: 8/9/2005
Citation: Arakane, Y., Muthukrishnan, S., Beeman, R.W., Kanost, M.R., Kramer, K.J. 2005. Laccase 2 is the phenoloxidase gene required for beetle cuticle tanning. Proceedings of the National Academy of Sciences 102(32):11337-11342.

Interpretive Summary: Tanning of the insect exoskeleton hardens and darkens this structure, and gives it both strength and elasticity. Two kinds of phenoloxidase enzymes, laccase and tyrosinase, have been proposed to participate in tanning, but no one has been able to prove which enzyme is responsible for this process. We isolated several laccases and tyrosinases from the red flour beetle, Tribolium castaneum, and showed which ones were needed at various stages of development. To establish the involvement of these types of genes in cuticle tanning, we blocked the activity of each one (one-at-a-time) by injection of gene-specific inhibitors called “dsRNAs”. No effect on the exoskeleton was observed after specific gene-knockout, with the single exception of a gene called laccase-2. dsRNA targeted to laccase-2 was followed by failure of the new exoskeleton to tan, and the insects remained soft-bodied and colorless, became deformed, and subsequently died in a dsRNA dose-dependent fashion. The results support the hypothesis that laccase-2 is the only phenoloxidase enzyme needed for cuticle tanning in Tribolium. Now that the exoskeleton tanning enzyme has been identified, it can become a target in screening assays for new biopesticides that weaken the protective outer coat of the insect.

Technical Abstract: Cuticle tanning or sclerotization involves the oxidative conjugation of proteins, which renders them insoluble and also hardens and darkens the color of the exoskeleton. Two kinds of phenoloxidases, laccase and tyrosinase, have been proposed to participate in tanning, but evidence regarding the identity of the enzyme(s) responsible for this process has been inconclusive. We report the cloning of cDNAs for laccases and tyrosinases from the red flour beetle, Tribolium castaneum, as well as their developmental patterns of expression. To establish the involvement of these types of enzymes in cuticle tanning, we performed dsRNA-mediated RNAi experiments to decrease expression of individual phenoloxidases. Normal phenotypes were obtained in every case except for laccase 2 dsRNA-treated insects that failed to tan, were soft-bodied, deformed, and subsequently died in a dsRNA dose-dependent fashion. The results support the hypothesis that two isoforms of laccase 2 generated by alternative splicing catalyze cuticle tanning in Tribolium.