Essential roles of LmKnk and LmRtv in chitin formation during locust pro-nymphal cuticle development

The pro-nymphal stage is critical for cuticle formation in the embryonic development of the migratory locust, Locusta migratoria, providing essential protection for embryonic stability and hatching. However, the molecular mechanisms regulating cuticular lamellar assembly during this stage remain largely unknown, especially in hemimetabolous insects. This study focuses on the roles of Knickkopf (LmKnk) and Retroactive (LmRtv) in the formation of the lamellar cuticle in the pro-nymphal stage. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis showed that transcript levels of LmKnk and LmRtv were significantly upregulated during the key cuticle formation period (embryonic day 8 to day 11, E8 to E11). RNA interference-mediated silencing of either gene resulted in a significantly reduced hatching rate, with over half of the individuals dying due to molting failure; disrupted the cuticular ultrastructure, including complete loss of the typical chitin lamellar structure; and impaired the cuticular barrier function, resulting in severely reduced desiccation tolerance. Immunofluorescence localization showed LmRtv is distributed in epidermal cells, suggesting a potential role in facilitating LmKnk trafficking to the procuticle. LmKnk then integrates into the cuticular matrix to ensure ordered chitin assembly. This study is the first to demonstrate the essential role of the LmKnk and LmRtv are essential for embryonic cuticle formation in a hemimetabolous insect, providing mechanistic insights into structural regulation and identifying a potential target for developing cuticle-based pest control strategies.