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Title: Chymotrypsin-like peptidases from Tribolium castaneum: A role in molting revealed by RNA interference

Author
item BROEHAN, GUNNAR - University Of Osnabruck
item ARAKANE, YASUYUKI - Kansas State University
item Beeman, Richard
item KRAMER, KARL - Collaborator
item MUTHUKRISHNAN, SUBBARATNAM - Kansas State University
item MERZENDORFER, HANS - University Of Osnabruck

Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/31/2009
Publication Date: 3/1/2010
Citation: Broehan, G., Arakane, Y., Beeman, R.W., Kramer, K.J., Muthukrishnan, S., Merzendorfer, H. 2010. Chymotrypsin-like peptidases from Tribolium castaneum: A role in molting revealed by RNA interference. Insect Biochemistry and Molecular Biology. 40(3):274-283. doi:10.1016/j.ibmb.2009.10.009.

Interpretive Summary: Digestion and exoskeleton are unique and sensitive aspects of insect physiology that could be exploited by appropriately-targeted biopesticides. Chymotrypsins are a small but important group of digestive enzymes that have been suspected to have an additional role in synthesis of the exoskeleton. In this work we confirmed for the first time that two chymotrypsins do indeed have vital and specific functions in insect exoskeleton and molting, and that disruption of these genes prevents the insect from shedding the old skin, resulting in death. Studying the detailed functions of insect genes continues to reveal new weaknesses that may be exploited for insect control.

Technical Abstract: Chymotrypsin-like peptidases (CTLPs) of insects are primarily secreted into the gut lumen where they act as digestive enzymes. We studied the gene family encoding CTLPs in the genome of the red flour beetle, Tribolium castaneum. Using an extended search pattern, we identified 14 TcCTLP genes that encode peptidases with S1 specificity pocket residues typically found in chymotrypsin-like enzymes. We further analyzed the expression patterns of seven TcCTLP genes at various developmental stages. While some TcCTLP genes were exclusively expressed in feeding larval and adult stages (TcCTLP-5A/B, TcCTLP-6A), others were also detected in non-feeding embryonic (TcCTLP-5C, TcCTLP-6D) and pupal stages (TcCTLP-5C, TcCTLP-6C/D/E). TcCTLP genes were expressed predominantly in the midgut, where they presumably function in digestion. However, TcCTLP-6C and TcCTLP-5C also showed considerable expression in the carcass. The latter two genes might therefore encode peptidases that act as molting fluid enzymes. To test this hypothesis, we performed western blots using protein extracts from larval exuviae. The extracts reacted with antibodies to TcCTLP-5C and TcCTLP-6E suggesting that the corresponding peptidases are secreted into the molting fluid. Finally, we performed systemic RNAi experiments. While injections of five TcCTLP-dsRNAs into penultimate larvae did not affect growth or development, injection of dsRNA for TcCTLP-5C and TcCTLP-6C resulted in severe molting defects.