Two integument-enriched chemosensory proteins contribute to deltamethrin tolerance through cuticular binding in...

Insect chemosensory proteins (CSPs) are ubiquitously distributed in both chemosensory and non-chemosensory tissues and play multiple physiological roles. Recent studies have demonstrated that CSPs-mediated resistance/tolerance is a novel mechanism of insecticide resistance/tolerance. Two CSP genes (LmCSP3 and LmCSP4) in Locusta migratoria could be induced by deltamethrin. However, it remains unclear whether they function in deltamethrin tolerance. Thus, in this study, we investigated the structural characteristics, spatiotemporal expression patterns, and functional roles of LmCSP3 and LmCSP4 in deltamethrin tolerance. The results showed that the two LmCSPs include the conserved olfactory-specific protein D (OS-D) domain and four invariant cysteine residues arranged in a C1-X8-C2-X18-C3-X2-C4 pattern. Both LmCSPs showed integument-enriched expression patterns. LmCSP3's peak expression was found in the 5th-instar nymphs while LmCSP4's expression was observed in the 3rd and 5th instars. RNAi-mediated knockdown of LmCSP3 and LmCSP4 increased locust mortality by 20.86% and 25.96% at a diagnostic deltamethrin concentration of 18 μg/mL, respectively (P ≤ 0.05). HPLC-based quantification demonstrated that silencing of LmCSP3 and LmCSP4 significantly increased the cuticular penetration rate of deltamethrin by 20.15% and 22.16%, respectively (P ≤ 0.01). In vitro fluorescence competitive binding assays showed that LmCSP3 had moderate binding affinities to deltamethrin while LmCSP4 exhibited weaker interaction to deltamethrin. These results suggested that locust cuticle LmCSP3 and LmCSP4 contributed to the deltamethrin tolerance by binding the insecticides within the cuticular matrix. Our findings elucidate the association between CSPs and deltamethrin tolerance in L. migratoria, providing critical insights for targeted resistance management strategies.