Functional analysis of the Tribolium castaneum Knickkopf gene in organization of cuticle

The insect cuticle is mainly composed of chitin and proteins, and forms a protective barrier that shields insects against biological and mechanical stresses. Proteins associated with chitin metabolism and/or cuticle assembly are attractive targets for biopesticide development. Recent studies of Drosophila melanogaster (Dm) have identified a gene, knickkopf, whose expression is important for tracheal tube expansion and cuticle organization. In the present study, we have identified three Knk-like genes in Tribolium castaneum, TcKnk1, TcKnk2 and TcKnk3, which are orthologs of DmKnk1, DmKnk2 and DmKnk3, respectively. All of these genes were differentially expressed during different developmental stages of the beetle, suggestive of distinct roles for each TcKnk in Tribolium development. All three Knk genes are expressed in the carcass (whole body without gut) but not in gut tissue. RNA interference (RNAi) of TcKnk1 resulted in lethal phenotypes at larval-larval, larval-pupal and pupal-adult molts, whereas RNAi of TcKnk2 and TcKnk3 led to developmental arrest only at the pupal-adult molt with ~55% and 100% mortality, respectively. Confocal microscopic analysis of epidermal tissues indicated co-localization of TcKnk1 with cuticular chitin in control insects. A significant reduction in chitin content was observed after RNAi for TcKnk1. Transmission electron microscopic analysis confirmed that TcKnk1-specific RNAi lethality is associated with defects in cuticle organization or synthesis. Collectively, our results suggest that Knks play crucial roles in the synthesis and/or organization of chitin in the cuticle of the red flour beetle and other species of insects.