A dual-barrier disruption strategy employing lufenuron/dsRNA nanocomplexes overcomes both cuticular and intestinal barriers in late-instar lepidopteran pests.

Managing late-instar lepidopteran pests remains a critical challenge due to their robust physiological and physical barriers, which severely compromise the efficacy of insecticides. Lufenuron, an insect growth regulator, controls lepidopteran pests by inhibiting chitin biosynthesis, but its efficacy against late-instar larvae needs improvement. Here, we developed a dual-barrier disruption strategy by integrating nano-lufenuron with RNA interference targeting the chitin synthase genes PxCHS1 and PxCHS2 in the diamondback moth, Plutella xylostella. The nanocarrier star polycation (SPc)-mediated lufenuron enhanced both stomach and contact toxicity to the diamondback moth larvae, and induced high expression of PxCHS1 and PxCHS2. PxCHS1 and PxCHS2 were predominantly expressed in the integument and midgut peritrophic matrix, respectively. SPc-mediated RNA interference of PxCHS1 caused cuticle thinning and enhanced the contact toxicity of nano-lufenuron, whereas silencing of PxCHS2 disrupted peritrophic matrix integrity and increased stomach toxicity. Furthermore, dsPxCHS1 and dsPxCHS2 self-assembled with the lufenuron/SPc nanocomplex, facilitating coordinated penetration and uptake across both cuticular and intestinal barriers. Co-delivery of LUF/SPc/dsPxCHS1 + dsPxCHS2 significantly increased mortality and reduced adult emergence in late-instar larvae. Collectively, this study establishes a barrier-targeted nanopesticide framework for overcoming the physical barriers and provides a novel dual-barrier-targeted nanopesticide strategy for efficient control of late-instar diamondback moth larvae, with potential reference for other lepidopteran pests.