|CHAUDHARI, SUJATA - Kansas State University
|ARAKANE, YASUYUKI - Chonnam National University
|SPECHT, CHARLES - University Of Massachusetts
|MOUSSIAN, BERNARD - University Of Tubingen
|KRAMER, KARL - Retired ARS Employee
|MUTHUKRISHNAN, SUBBARATNAM - Kansas State University
|BEEMAN, RICHARD - Retired ARS Employee
Submitted to: Developmental Cell
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/7/2012
Publication Date: 1/31/2013
Citation: Chaudhari, S.S., Arakane, Y., Specht, C.A., Moussian, B., Kramer, K.J., Muthukrishnan, S., Beeman, R.W. 2013. Retroactive maintains cuticle integrity by promoting the trafficking of Knickkopf into the procuticle of Tribolium castaneum. Developmental Cell. 9(1):e1003268. DOI: http://dx.doi.org/10.1371/journal.pgen.1003268.
Interpretive Summary: Insects must periodically replace their protective, inelastic exoskeleton as they grow. This complex, delicate, and intricate process is an attractive target for intervention with a biopesticide, but is still not well understood. We recently showed that the replacement exoskeleton is shielded from collateral damage during degradation and recycling of the overlying old exoskeleton by a protective protein called “Knickkopf”. In the present work, we show that Knickkopf requires the presence of a second protein called “Retroactive”, and that this latter protein probably acts by chaperoning Knickkopf to its proper extracellular location in the new exoskeleton. Identification of all the protein components of exoskeletal synthesis and recylcing will enable us to design novel biopesticides that target the most sensitive aspects of insect development.
Technical Abstract: Molting, or the replacement of the old exoskeleton with a new cuticle, is a complex developmental process that all insects must undergo to maintain growth. Our work has uncovered a major pathway involved in cuticular chitin maintenance wherein accumulation of the Tribolium castaneum knickkopf protein (TcKnk) in the growing chitinous matrix of the procuticle facilitates chitin organization and confers protection from chitinolytic enzymes. Deletion mutants in either of two genes, knickkopf (knk) or retroactive (rtv), were shown to exhibit cuticular defects and embryonic lethality in Drosophila melanogaster. Both, knk and rtv mutant embryos exhibited “blimp” phenotypes similar to krotzkopf verkehrt (kkv) mutants (an ortholog of Tribolium chitin synthase A), indicating their role in maintenance of cuticle integrity. We have detected an ortholog of Drosophila Rtv in the T. castaneum genome, and have named it TcRtv. Confocal microscopic analysis revealed localization of TcRtv protein predominantly inside the epidermal cells. RNA interference (RNAi) of TcRtv resulted in molting defects and a chitin depletion phenotype similar to those following TcKnk and TcChs-A dsRNA treatment. Co-injection of dsRNAs for TcRtv and TcCht-5 rescued the chitin depletion phenotype, indicating a role for TcRtv in protecting chitin from chitinases. In the absence of TcRtv, TcKnk protein was mislocalized within the epithelial cells (instead of the procuticle), indicating an important role for TcRtv in proper trafficking of TcKnk to the procuticle where the latter protein associates with and protects chitin. The data demonstrate that TcRtv indirectly promotes cuticle synthesis and organization by facilitating the movement and localization of Knk in the procuticle.