The excessive use of chemical pesticides brings many environmental problems. Botanical pesticides seem to be a good alternative to control pests, but their slow action and lack of residual action constrain their further application. Here, we developed a star polycation (SPc) as the drug nanocarrier that could combine matrine into a complex to decrease its particle size to the nanoscale. The combination of SPc and matrine increased the zeta potential and the noncovalent molecular interaction, such as hydrogen bonds and hydrophobic interactions playing an important role in the complex formation. The nanosized matrine/SPc complex exhibited a higher cytotoxicity and an enhanced lethal effect on insect pests compared to those of matrine alone. As a botanical pesticide adjuvant, the SPc could increase the control efficacies of matrine, D-limonene, and pyrethrin against insect pests, achieving a great improvement in toxicity and persistence. The construction of SPc provides a powerful tool for delivering pesticides to increase their control efficacies and decrease their application amounts and rates, showing great potential for sustainable pest management.

Nanoparticles have been widely applied as gene carrier for improving RNA interference (RNAi) efficiency in medical and agricultural fields. However, the mechanism and delivery process of nanoparticle-mediated RNAi is not directly visualized and elucidated. Here we synthesized a star polymer (SPc) consisted of a hydrophilic shell with positively-charged tertiary amine in the side chain, which was taken as an example to investigate the mechanism in gene delivery. The SPc could assemble with dsRNA spontaneously through electrostatic force, hydrogen bond and van der Waals force. Interestingly, the SPc could protect dsRNA from degradation by RNase A and insect hemolymph, thus remarkably increasing the stability of dsRNA. Meanwhile, the SPc could efficiently promote the cellular uptake and endosomal escape for intracellular spreading of dsRNA. Transcriptome analysis revealed that the SPc could up-regulate some key genes such as Chc, AP2S1 and Arf1 for activating clathrin-mediated endocytosis. Furthermore, the suppression of endocytosis hindered the cellular uptake of SPc-delivered dsRNA in vitro, and the subsequent RNAi effect was also disappeared in vivo. To our knowledge, our study is the first direct visualization of the detailed cellular delivery process and mechanism of nanocarrier-mediated gene delivery. Above mechanism supports the application of nanocarrier-based RNAi in gene therapy and pest management. Graphical Abstract

There are two main limitations for sprayable RNA pesticide development: delivery e ciency and synthetic cost of double-stranded RNA (dsRNA). We previously constructed a nanocarrier-based transdermal dsRNA delivery system and a novel bacteria-based hairpin RNA (hpRNA) expression system to solve these challenges. Herein, as a subsequent exploration of RNA pesticide (sprayable ds/hpRNA for pest control), we performed a greenhouse application of bacteria-expressed and nanocarrier-delivered RNA pesticide on green peach aphid. The nanoparticle SPc could combine and deliver dsRNA across the aphid cuticle, and V-type proton ATPase subunits d (ATP-d) and G (ATP-G) were selected as the potential RNA interference (RNAi) targets. Our plasmid-Escherichia coli system simultaneously expressing ATP-d and ATP-G hairpin RNAs (hpRNAs) was constructed for mass production of hpRNA. The expressed hpRNA was mixed with SPc and detergent to form RNA formulation, which showed a certain insecticidal activity through the spray application in the greenhouse. Total control e cacy of our RNA pesticide could reach 61% on 3 d, and maintained at 50% until the sixth day. To our knowledge, our study is the rst attempt to apply the bacteria-expressed and nanocarrier-delivered RNA pesticides for pest control in the greenhouse trial, which is bene cial for promoting the development of RNA pesticides. Key Message• SPc can protect and deliver dsRNA from degradation for e cient gene silencing• hpRNA is expressed in E. coli system to develop a novel RNA insecticide • Our RNA pesticide can be applied to control aphids in greenhouse • Our study brings the concept of RNAi-based pest management to practical application

RNA interference (RNAi) has developed rapidly as a potential “green” pest management strategy. At present, most studies have focused on the screening of aphid lethal genes, whereas only a few studies have been conducted on wing development, which is crucial for aphid migration and plant‐virus dissemination. Here, the Myzus persicae genes vestigial (vg) and Ultrabithorax (Ubx) related to wing development, were cloned. These two genes were expressed in various tissues of 3rd‐instar winged aphids. The mRNA level of vg was high in 3rd‐instar nymphs, whereas the expression level of Ubx was high in adults. The nanocarrier‐mediated delivery system delivered double‐stranded RNAs for aphid RNAi using topical and root applications. The expression levels of vg and Ubx were downregulated by 44.0% and 36.5%, respectively, using the topical application. The simultaneous RNAi of the two target genes caused 63.3% and 32.2% wing aberration rates using topical and root applications, respectively. The current study provided a promising method for controlling aphid migration to alleviate the spread of insect transmitted plant diseases.

RNA interference (RNAi) has become an essential technique in functional analysis of insect genes and pest management. However, insensitivity to RNAi is the main obstacle that limits the identification of functional genes in lepidopteran insects. Hereby we explored an efficient nanocarrier (star polycation, SPc)-mediated dsRNA delivery system to achieve efficient RNAi at all developmental stages of lepidopteran insect Spodoptera frugiperda. Double-stranded RNA (dsRNA) was synthesized targeting a consistently expressed gene V-type proton ATPase subunit d (ATP-d) as well as enhanced green fluorescent protein (eGFP) for various operation tests. At the egg and early larval stages, SPc-loaded dsRNA could penetrate the physical obstacles of eggshell and larval body wall. Thus, soaking and topical applications of SPc-loaded dsRNA were suitable for efficient RNAi at egg and 1st-2nd instar larval stages, respectively. The SPc could protect dsRNA from degradation by midgut fluid of mid-late instar larvae. Thereby oral feeding of SPc-loaded dsRNA silenced the target gene at mid-late larval stages more efficiently compared to dsRNA alone. Finally, for pupal and adult stages, injection of SPc-loaded dsRNA was enough to induce high RNAi effect. A key developmental gene, Chitin deacetylase 1 (CDA1), was taken as an example to test the pest control efficiency of SPc-based RNAi system. The constructed system could remarkably induce the knockdown of CDA1 and lead to growth retardation and mortality. Overall, the present study constructed an efficient nanocarrier-based RNAi system for whole developmental stages of lepidopteran insects, which would provide a powerful tool for identifying functional genes in lepidopteran insects.