Functional analysis of the knickkopf gene gamily in organization of Tribolium castaneum cuticle

The insect cuticle is composed mainly of chitin and proteins and is a protective barrier that shields insects against biological and mechanical stresses. Proteins associated with chitin metabolism and cuticle assembly are attractive targets for biopesticide development. Recent studies in Drosophila (Dm) have identified a gene, knickkopf (knk), whose expression is important for tracheal tube expansion and cuticle organization. In the present study, we have identified three Knk-like genes in Tribolium, TcKnk1, TcKnk2 and TcKnk3 which are orthologs of DmKnk1, DmKnk2 and DmKnk3, respectively. All of these genes were differentially expressed throughout the different developmental stages of the beetle, suggestive of crucial roles for TcKnk’s in Tribolium development. All three genes are expressed in the carcass (whole body without gut) but not in gut tissue. RNAi of TcKnk1 resulted in lethal phenotypes preventing larval-larval, larval-pupal and pupal-adult molts, whereas RNAi of TcKnk2 and TcKnk3 resulted in developmental arrest only at the pupal-adult molt with ~55% and 100% mortality, respectively. Interestingly, lethal phenotype was observed only with dsRNAs specific for the C-terminal region of TcKnk3 but not its N-terminal region, suggestive of alternative sliced variants of TcKnk3. TEM analysis confirmed thatTcKnk1-specific RNAi lethality is associated with defects in cuticle organization. Collectively, our results indicate that each Knk plays a different role in the synthesis or organization of chitin in the cuticle of Tribolium.