Cuticle thickening mediates insecticide penetration resistance in Spodoptera litura

INTRODUCTION: Long-term and extensive use of chemical pesticides has led to the development of resistance in many important agricultural pests. The mechanisms of resistance formation in pests are complex and variable, and unraveling the resistance mechanisms is the key to control resistant pests. Insect cuticle, as the first line of defense for insecticides, plays a non-negligible role in insecticide penetration resistance. Although penetration resistance is widespread in insects, the multiple molecular mechanisms that impede insecticide penetration are unclear, especially in Spodoptera litura. OBJECTIVES: This study aims to reveal the molecular mechanisms of insecticide penetration resistance in S. litura. METHODS: The structure and thickness of cuticle were analyzed by TEM, and the role of cuticle in penetration resistance was determined by different application methods. The molecular mechanism of cuticular proteins overexpression was analyzed using RNAi, TEM, dual-luciferase assay and EMSA from cis- and trans-acting factors. In addition, the relationship between the chitin synthetic pathway and insecticide resistance was explored through enzyme activity, inhibitor assay, molecular docking and RNAi. Furthermore, the role of 20E in penetration resistance was analyzed. RESULTS: The cuticle of the resistant populations was significantly thickened and accompanied by extrusion, which contributed significantly to indoxacarb resistance. Constitutive upregulation of trans-acting factor SlituFTZ-F1 co-regulates the overexpression of SlituCP26 with cis-acting elements in the SlituCP26 promoter (74 bp insertion), affecting the cuticle thickness‑mediated indoxacarb penetration resistance. Meanwhile, the overexpression of key genes in the chitin synthesis pathway increased the chitin content, which combined with SlituCP26 to participate in indoxacarb resistance. Moreover, 20E affected the SlituFTZ-F1-mediated regulatory pathway and chitin biosynthesis pathway in indoxacarb resistance. CONCLUSION: This study comprehensively elucidated the molecular mechanism of cuticle thickening mediating penetration resistance to indoxacarb and confirmed its existence in the field populations of S. litura.