Identification and Functional Insights of Knickkopf Genes in the Larval Cuticle of Leptinotarsa decemlineata

The Colorado potato beetle (<i>Leptinotarsa decemlineata</i>) is a major pest of potato crops. While <i>Knickkopf</i> (<i>Knk</i>) genes are essential for insect cuticle formation, their roles in pests like <i>L. decemlineata</i> remain unclear. This study aims to identify and characterize <i>Knk</i> genes in <i>L. decemlineata</i> and explore their functions in larval development and cuticle integrity. We used genomic and transcriptomic databases to identify <i>LdKnk</i>-family genes, validated through RT-PCR and RACE. Gene expression was analyzed at various developmental stages and tissues using qRT-PCR. RNA interference (RNAi) and Transmission electron microscopy (TEM) were applied to determine the functional roles of these genes. Four <i>LdKnk</i>-family genes were identified. Spatio-temporal expression analysis indicated significant gene expression during larval molting and pupal stages, especially in the epidermis. RNAi experiments showed that silencing <i>LdKnk</i> and <i>LdKnk3-5</i>' led to reduced larval weight, cuticle thinning, and increased mortality, while <i>LdKnk3-FL</i> knockdown caused abnormal cuticle thickening and molting disruptions. <i>LdKnk2</i> knockdown increased epicuticle and endocuticle thickness without visible phenotypic changes. The study highlights the essential roles of <i>LdKnk</i>-family genes in maintaining cuticle structure and integrity, suggesting their potential as targets for RNAi-based pest control.