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

The Colorado potato beetle (Leptinotarsa decemlineata) is a major pest of potato crops. While Knickkopf (Knk) genes are essential for insect cuticle formation, their roles in pests like L. decemlineata remain unclear. This study aims to identify and characterize Knk genes in L. decemlineata and explore their functions in larval development and cuticle integrity. We used genomic and transcriptomic databases to identify LdKnk-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 LdKnk-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 LdKnk and LdKnk3-5' led to reduced larval weight, cuticle thinning, and increased mortality, while LdKnk3-FL knockdown caused abnormal cuticle thickening and molting disruptions. LdKnk2 knockdown increased epicuticle and endocuticle thickness without visible phenotypic changes. The study highlights the essential roles of LdKnk-family genes in maintaining cuticle structure and integrity, suggesting their potential as targets for RNAi-based pest control.

Crucial roles of specialized chitinases in elytral and hindwing cuticles construction in Leptinotarsa decemlineata.

BACKGROUND: The Colorado potato beetle (CPB), Leptinotarsa decemlineata, is a major potato (Solanum tuberosum) pest, infesting over 16 million km2 and causing substantial economic losses. The insect cuticle forms an apical extracellular matrix (ECM) envelope covering exposed organs to direct morphogenesis and confer structural protection. While select chitinase (Cht) genes have proven essential for larval development, their potential activities directing ECM remodeling underlying adult wing maturation remain undefined. RESULTS: We investigated the expression patterns and performed an oral RNA interference (RNAi) screen targeting 19 LdChts in late-instar L. decemlineata larvae. Subsequently, we assessed their effects on adult eclosion and wing characteristics. Knockdown of LdCht5, LdCht7, LdCht10, LdIDGF2, and LdIDGF4, as well as others from Group IV (LdCht15, LdCht12, LdCht17, and LdCht13) and Groups VII-X (LdCht2, LdCht11, LdCht1, and LdCht3), resulting in shrunken, misshapen elytra with reduced areal density, as well as transverse wrinkling and impaired wing-tip folding in hindwings. Scanning electron micrographs revealed eroded elytral ridges alongside thinned, ruptured hindwing veins, indicative of mechanical fragility post-LdCht suppression. Spectroscopic analysis uncovered biomolecular alterations underlying the elytral anomalies, including decreases in peaks representing chitin, proteins, and lipids. This loss of essential ECM components provides evidence for the fragility, wrinkling, and shrinkage observed in the RNAi groups. CONCLUSION: Our findings elucidate the crucial role of chitinases in the turnover of chitinous cuticles on beetle wings, offering insights into RNAi-based control strategies against this invasive pest. © 2024 Society of Chemical Industry.