Twinstar is a chitin synthase interacting protein with an essential role in insect cuticle biosynthesis

Chitin is an abundant natural biopolymer that plays a crucial role in insect growth and development as a fundamental structural component of the exoskeleton. The membrane-integral β-glycosyltransferase, chitin synthase, has been identified as the central component in chitin biosynthesis. However, the precise roles of other proteins in facilitating chitin synthase in chitin biosynthesis remain unclear. In this study, we employed split-ubiquitin membrane yeast two-hybrid (MYTH) and pull-down assays to demonstrate the physical interaction between Twinstar (Tsr), a small molecular protein in the actin-depolymerizing factor ADF/Cofilin protein family, and chitin synthase Krotzkopf verkehrt (Kkv) in Drosophila melanogaster in vitro. The RNA interference (RNAi)-mediated global knockdown of Tsr in D. melanogaster resulted in larval lethality. Furthermore, targeted suppression of Tsr in the tracheal and epidermal tissues also led to larval mortality, while knocking down Tsr in the wing tissues led to wrinkled wings. Additionally, silencing Tsr not only reduced the chitin content in the first longitudinal vein of the wings but also led to the absence of the chitin lamellar structure. To validate the functional conservation of Tsr in other insect orders, the two agricultural pests Ostrinia furnacalis and Tribolium castaneum, representing lepidoptera and coleoptera insects, respectively, were investigated. Knockdown experiments targeting the Drosophila Tsr orthologues OfTsr in O. furnacalis and TcTsr in T. castaneum produced abnormal larvae during molting or pupation in O. furnacalis and lethality in T. castaneum. Our findings not only improve our knowledge of the chitin biosynthesis machinery in insect cuticles but also provide new potential targets for the control of major agricultural pests.