Nanocarrier-delivered dsRNA targeting cuticular protein CPAP3-2 enhances Etoxazole efficacy in tetranychid mites

Panonychus citri is a major citrus pest causing significant yield losses. Prolonged use of acaricides like etoxazole, a diphenyloxazoline chitin synthesis inhibitor, has led to resistance and environmental concerns, necessitating novel control strategies. This study presents a dsRNA delivery approach utilizing graphene oxide nanoparticles (GONs), designed to decrease acaricide application while increasing control efficacy against tetranychidae. First, the study found that etoxazole is highly lethal to the active developmental stages of P. citri, by disrupting chitin metabolism and causing fatal molting abnormalities. Moreover, etoxazole treatment significantly upregulates PcCPAP3-2, a gene essential for cuticle integrity, while silencing this gene via RNAi not only made the mites' cuticles more susceptible to damage but also significantly increased their sensitivity to etoxazole. To enhance the stability and efficacy of dsRNA, the study employed an E. coli expression system to produce large quantities of dsPcCPAP3-2. GONs were used as a delivery vehicle to facilitate dsRNA stability and functionality. Experimental results confirmed that GONs effectively protected dsRNA from environmental degradation and promoted its functional expression within P. citri. The etoxazole/GONs-dsPcCPAP3-2 complex exhibited the highest mortality rate, reducing nymph survival to 23.74%. Tetranychus urticae shares similar resistance challenges. Homology analysis revealed that PcCPAP3-2 shares 83.75% nucleotide identity with its T. urticae ortholog. GONs-dsPcCPAP3-2 effectively silenced TuCPAP3-2, reduced oviposition, and significantly increased mortality when co-applied with etoxazole. Conclusively, this study demonstrates that GONs-mediated targeted dsRNA delivery significantly enhances P. citri sensitivity to acaricides, offering a promising strategy to combat resistance and reduce pesticide use.