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Title: Molecular and functional analyses of amino acid decarboxylases involved in cuticle tanning in Tribolium castaneum

Author
item ARAKANE, YASUYUKI - KANSAS STATE UNIVERSITY
item LOMAKIN, JOSEPH - UNIVERSITY OF KANSAS
item Beeman, Richard
item MUTHUKRISHNAN, SUBBARATNAM - KANSAS STATE UNIVERSITY
item GEHRKE, STEVIN - UNIVERSITY OF KANSAS
item KANOST, MICHAEL - KANSAS STATE UNIVERSITY
item KRAMER, KARL - 5430-05-30 COLLABORATOR

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/17/2009
Publication Date: 6/12/2009
Citation: Arakane, Y., Lomakin, J., Beeman, R.W., Muthukrishnan, S., Gehrke, S.H., Kanost, M.R., Kramer, K.J. 2009. Molecular and Functional Analyses of Amino Acid Decarboxylases Involved in Cuticle Tanning in Tribolium castaneum. Journal of Biological Chemistry 284:16584-16594.

Interpretive Summary: The exoskeleton forms a protective outer covering or “skin” of insects, and is essential for movement, resistance to infection, and mediating oxygen exchange. We have recently characterized the roles of several components involved in the hardening and darkening of the exoskeleton after the insect sheds its old skin. By inhibiting the normal functioning of these intermediates, we showed that they were needed to confer the proper hardness, flexibility and color of the mature exoskeleton. Studies such as these will lead to better understanding of insect growth and development and better strategies for disrupting the associated genes for pest control.

Technical Abstract: Aspartate 1-decarboxylase (ADC) and dopa decarboxylase (DDC) provide b–alanine and dopamine used in insect cuticle tanning. Beta-alanine is conjugated with dopamine to yield N-b-alanyldopamine (NBAD), a substrate for the phenoloxidase laccase that catalyzes the synthesis of cuticle protein cross-linking agents and pigment precursors. We identified ADC and DDC genes in the red flour beetle, Tribolium castaneum (Tc), and investigated their functions. TcADC mRNA was most abundant prior to the pupal-adult molt. Injection of TcADC dsRNA (dsTcADC) into mature larvae resulted in depletion of NBAD in pharate adults, accumulation of dopamine, and abnormally dark pigmentation of the adult cuticle. A similar pattern of catechol content consisting of elevated dopamine and depressed NBAD was observed in the genetic black mutants of Tribolium, in which levels of TcADC mRNA are drastically reduced. In addition, cuticles from the Tribolium black mutant and dsTcADC-injected insects both exhibited similar changes in material properties. Dynamic mechanical analysis of elytral cuticle from beetles with depleted TcADC transcripts revealed diminished cross-linking of cuticular components, further confirming the important role of oxidation products of NBAD as cross-linking agents during cuticle tanning. Injection of dsTcDDC into larvae produced a lethal pupal phenotype and the resulting grayish pupal cuticle exhibited many small patches of black pigmentation. When dsTcDDC was injected into young pupae, the resulting adults had abnormally dark-brown body-color, but there was little mortality. Injection of dsTcDDC resulted in more than a five-fold increase in levels of DOPA, indicating that lack of TcDDC led to accumulation of its substrate, DOPA.