The full-length cDNA sequences encoding JHEH, JHDK and MFE were obtained from the CBW transcriptome. The presence of domains and motifs typically found in these proteins was confirmed through multiple alignments of the predicted proteins with homologous sequences available in GenBank. The alignment was performed using the Clustal Omega algorithm (https://www.ebi.ac.uk/Tools/msa/clustalo/). The dsRNA molecules were designed on the basis of the coding DNA sequence (CDS) of each gene using the E-RNAi web tool (https://www.dkfz.de/signaling/e-rnai3/). To mitigate possible off-target effects, a cutoff of <19 bp homology for putative sequences from honeybee (Apis mellifera) and fruit fly (Drosophila melanogaster) was used in the design of the dsRNAs. All sequence details and sources are described in Supplementary Data S1. Template DNA plasmids (pCloneEZ-NRS-Blunt-Amp) containing the partial sequences of the target genes flanked by the T7 promoter were purchased from Epoch Biolabs Inc. (Texas, USA). The vectors were transformed into chemically competent OmniMAX E. coli. Then, the plasmid DNA was isolated by alkaline lysis (Ehrt and Schnappinger, 2003) and used as a template for the PCR amplification of each gene fragment using specific primers (Supplementary Table S1). The PCR product was purified using a PureLink™ PCR Purification Kit according to manufacturer’s instructions (Invitrogen, Massachusetts, United States). Using 8 µL (250 ng/μL) of the purified PCR product as the template, dsRNA synthesis was performed in reactions of 20 µL using a MEGAscript T7 RNAi Kit (Invitrogen, Massachusetts, United States). The transcription reaction was run overnight and then purified according to the manufacturer’s instructions.

The CS-TPP-dsRNA complexes were assembled as described by Dhandapani et al. (2019) with slight modifications. Briefly, low-molecular-weight CS (85% deacetylated) (Sigma‒Aldrich, Darmstadt, Germany) was dissolved in 1% acetic acid (2 mg/mL), and sodium tripolyphosphate (TPP) (Sigma‒Aldrich, Darmstadt, Germany) was dissolved in Milli-Q ultrapure water (2.5 mg/mL). Then, 2 mL of dsRNA (2.5 mg/mL) and 2 mL of TPP (2.5 mg/mL) were added dropwise to 5 mL of CS solution under magnetic stirring, and the solution was stirred for 60 min at room temperature. The CS-TPP-dsRNA complex was prepared using a mass ratio of 5:10:1. Linear PEI 20 KDa (Invitrogen, Missouri, United States) was dissolved in Milli-Q ultrapure water (1 mg/mL). Thereafter, the dsRNA-PEI complexes were prepared by mixing 6 mL of Milli-Q ultrapure water, 2 mL of dsRNA (2.5 mg/mL), and 2 mL of PEI (1 mg/mL) under magnetic stirring for 60 min at room temperature. The dsRNA-PEI complex was prepared using a nitrogen:phosphate ratio of 6:1. Finally, both complexes were placed in an ultrasonic bath for 15 min. The final concentration of dsRNA in CS-TPP and PEI complexes was 500 ng/μL. Apart from the CS:dsRNA and PEI:dsRNA ratios mentioned above, 1:1 and 10:1 CS:dsRNA, as well as 1:1, 3:1, 10:1, and 20:1 N:P ratios (for PEI:dsRNA), were also formulated keeping the dsRNA and TPP amounts constant.

The CS-TPP-dsRNA and PEI-dsRNA complexes were characterized by dynamic light scattering (DLS) to determine their mean diameter (z-average), surface charge (zeta potential), and polydispersity (PdI). DLS measurements were performed using a Malvern Zetasizer Nano ZS (Malvern Panalytical, Worcestershire, United Kingdom) with three readings taken per sample with the following parameters: 25°C, material RI of 1.59, and dispersant (Milli-Q ultrapure water) RI and viscosity of 1.33 and 0.887, respectively. Samples for the DLS analysis were diluted to 50 ng/μL of dsRNA.

CBWs were obtained from the insect rearing platform of Embrapa Genetic Resources and Biotechnology (Brasília, DF, Brazil). The weevils were reared on an artificial diet (Supplementary Table S2) and maintained under standard rearing conditions of 28°C, 70% ± 5% relative humidity, and a 12:12 h light:dark photoperiod.

To perform the dsRNA degradation assay, hemolymph and gut fluid were extracted from third-instar CBW larvae, as described by Cooper et al. (2020). Briefly, the hemolymph was extracted from 10 individuals by performing a small incision on the dorsal side of the abdomen, and the larvae were allowed to bleed out on ice-cold PARAFILM (Sigma‒Aldrich, Missouri, United States). Then, the hemolymph was collected using a micropipette and placed into a tube containing 50 mg of phenylthiourea (Sigma‒Aldrich, Missouri, United States). For gut juice extraction, 10 individuals were gently held with tweezers by the anterior part of the body. Then, a gentle pressure was applied longitudinally in the larvae to stimulate peristaltic movements across the gut. After emesis, the gut juice was collected into a tube and centrifuged at 14,000g for 20 min at 4°C, and the supernatant was transferred to another tube. Protein contents from the hemolymph and gut juice were determined by fluorometry using a Quibit Protein Assay Kit in a Qubit 4 Fluorometer (Invitrogen, Massachusetts, United States). Then, the extracted hemolymph and gut fluid were diluted using PBS buffer to 500 ng/μL for an ex vivo assay.

For the ex vivo assay, 2 µL (250 ng/μL) of naked dsGFP (400 bp) and dsGFP complexed with CS-TPP or PEI were mixed with 3 µL (1.5 µg) of the hemolymph or gut fluids and 15 µL of 1× PBS. The pH of PBS was adjusted according to the pH of insect fluids (pH = 7.0 for hemolymph and pH = 6.0 for gut fluid). Control treatments included these mixtures without dsRNA or body fluids. The samples were incubated at 37°C for 30 min. After incubation, 2 μL of 6× Gel Loading Buffer (Thermo Fisher, Massachusetts, United States) was added to the samples, after which they were loaded onto a 1.5% agarose gel. Target dsRNA bands were visualized and photographed using a Gel Doc EZ Gel Documentation System (Bio-Rad, California, United States).

To evaluate the endogenous expression profiles of JHEH, JHDK, and MFE genes throughout the developmental stages of the CBW, three biological replicates were collected from each developmental stage. Each biological replicate was collected by pooling 15 first-instar larvae, 8 second-instar larvae, or 3 individuals from the other developmental stages (∼100 mg of tissue per replicate). Additionally, to compare the expression of JHEH, JHDK, and MFE between the carcass and gut tissues, tissue samples were dissected from third-instar larvae (five larvae per replicate, three replicates). All samples were frozen in liquid nitrogen and stored at −80°C for further RNA extraction, cDNA synthesis, and gene expression analysis.

Third-instar CBW larvae were dorsally injected with 2 µL of naked dsRNA, dsRNA + CS-TPP, or dsRNA + PEI. Three doses of dsMFE, dsJHEH, and dsJHDK were tested (0.05, 0.5, and 2 µg, respectively). Milli-Q ultrapure water (water, water + PEI, and water + CS-TPP), dsGFP, dsGFP + PEI, and dsGFP + CS-TPP were used as negative control treatments. The amount of naked dsGFP or dsGFP complexed with the nanoparticles injected into the insects was 2 µg. To evaluate gene silencing efficiency, three biological replicates were collected from each treatment 48 h after injection, immediately frozen in liquid nitrogen, and stored at −80°C until RNA extraction. Each biological replicate was formed by pooling three individuals. Once the optimal amount of dsRNA and the best type of nanoparticle for inducing silencing of the target genes in the CBW larvae were determined, further bioassays were performed to evaluate the effect of naked dsRNA and dsRNA-PEI on insect survival, phenotype, and gene silencing. In addition to the single dsRNA treatment, combinations of two dsRNAs were tested to evaluate whether the simultaneous silencing of multiple target genes increased insect mortality compared with the single-target strategy. The tested combinations of dsRNAs included dsMFE/JHDK, dsMFE/JHEH, and dsJHDK/JHEH. Third-instar larvae were injected with 500 ng of naked dsRNA, dsRNA-PEI, or a combination of the two types of dsRNAs (250 ng of each). The bioassays were repeated three times under the same conditions using 20 larvae per treatment (N = 60). For gene expression analyses, 20 larvae were injected with the tested treatments, and three biological replicates of three larvae were collected per treatment 48 h after injection. As a control group for each experiment, larvae were injected with the same amount of dsGFP. The injections were performed using a 700 Series Hamilton syringe (10 µL) (Hamilton Company, Nevada, United States) coupled with a 51-mm gauge 26S 4, 12° needle (Allcrom, São Paulo, Brazil).

The artificial diet was mixed with dsGFP, dsJHEH/JHDK, dsGFP + PEI, or dsJHEH/JHDK + PEI at two concentrations (100 and 1,000 ng of dsRNA per gram of diet). A total volume of 400 μL of the treated diet was applied to each microplate well (Thermo Fisher, Massachusetts, United States), and one newly hatched CBW larva was placed into the well. The larvae were transferred to new plates with the treated diet every 3 days and then fed an untreated diet from Day 15. Bioassays were repeated three times under the same conditions using 20 larvae per treatment (N = 60). For gene expression analyses, 20 larvae were fed on the tested treatments, and 4 biological replicates of three larvae each were collected per treatment 10 days after feeding. Phenotypic abnormalities and survival were recorded every day for 20 days. The representative images of the morphological alterations observed in the CBW insects upon dsRNA treatment were captured using a Leica DFC310 FX digital camera attached to a Leica MZ10 F stereoscope (Leica Microsystems, Wetzlar, Germany).

Total RNA was isolated using TRIzol Reagent (Invitrogen, Massachusetts, United States) according to the manufacturer’s instructions. The integrity of the RNA samples was evaluated on a 1% (w/v) agarose gel through electrophoresis, and the RNA samples were quantified using a NanoDrop-2000 spectrophotometer (Thermo Fisher Scientific Inc., Massachusetts, United States). The RNA samples were treated with DNase I and RNase-Free (1 U/µL) (Invitrogen, Massachusetts, USA), and first-strand cDNA was synthesized from 2 µg of RNA using Oligo(dT)30 primers and M-MLV reverse transcriptase (Invitrogen, Massachusetts, United States) according to the manufacturer’s instructions.

Each RT-qPCR mixture contained 5 μL GoTaq qPCR Master Mix (Promega, Wisconsin, USA), 2 μL cDNA (diluted 20×), 2.6 μL nuclease-free water, and 0.4 μL of each forward and reverse primer (10 µm). AgRPS26 and AgRPS11 were used as reference genes. RT-qPCR was performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, California, United States) under the following conditions: initial denaturation at 95°C for 15 min, followed by 40 cycles of 95°C for 30 s, 60°C for 20 s, and 72°C for 30 s. Primer efficiency was calculated using the MINER software, and relative gene expression analysis was performed following the 2^−ΔΔCt method (Pfaffl, 2001) using the qbase⁺ software (Biogazelle, Gent, Belgium). The primers employed for the gene expression analysis are listed in Supplementary Table S1.

Survival curves were generated using Kaplan‒Meier estimators, and the log-rank test was applied for pairwise comparisons between treatments. Gene expression differences among treatments were assessed by using one-way ANOVA with multiple comparisons (Tukey’s HSD). All statistical analyses were performed using the SPSS Statistics 27 software (IBM).

The CBW full-length MFE, JHDK, and JHEH ORF’s (open reading frame) were 816, 1,371, and 615 bp, respectively. The analysis of the primary structure of each predicted protein revealed the presence of key signatures (Supplementary Figure S1). The conserved cytochrome P450 site located at 401–410 aa and a membrane anchor region at 5–22 aa were the main motifs found in the MFE sequence. Three GTP-binding motifs, DxN (40–43 aa), xxE (156–158 aa), and PGNFIFGx (194–201 aa), as well as three elongation factor hand motifs (calcium binding), were detected in the JHDK sequence. Last, the HGWP epoxide hydrolase domain (154–158 aa) and the catalytic triad (Asp225, Glu406, and His432) were identified in the JHEH sequence.

The mean particle size (d.nm) and surface charge, determined by DLS, varied widely between different proportions of CS:dsRNA and PEI:dsRNA (Table 1). For CS-TPP-dsRNA nanoparticles, increasing CS amounts led to smaller nanoparticles, while increasing PEI amounts in PEI-dsRNA complexes showed the opposite (Supplementary Figure S2). Regarding the distribution of particles, the smallest polydispersity coefficients (0.198 and 0.209) were detected for 5:1 and 6:1 (N:P) proportions of CS:dsRNA and PEI:dsRNA, respectively (Table 1). These proportions also presented the highest zeta potential (∼34 and ∼31 mV for CS nanoparticles and PEI nanoparticles, respectively), and no further increase was detected with higher amounts of any polymer (Supplementary Figure S3). Owing to their low PdI and considering that higher proportions increased particle size but not surface charge, while lower proportions presented lower charge, CS:dsRNA 5:1 and PEI:dsRNA 6:1 (N:P) were selected as optimal proportions and used for downstream analysis (including bioassays) in our study.

Degradation of the naked dsRNA was not observed after incubation with the CBW hemolymph, while a complete degradation of the dsRNA was induced by the CBW gut juice. The dsRNA complexed with CS-TPP or PEI nanoparticles was protected from the nuclease activity of the gut juice. Some fraction of the dsRNA was released from CS-TPP when incubated with the hemolymph and gut juice, but dsRNA-PEI complexes were stable in both body fluids. In addition, naked dsRNAs and nanoparticle-complexed dsRNAs were both stable in the PBS solution, and no degradation of the dsRNA was detected (Figure 1).

The temporal and tissue expression profiles of MFE, JHEH, and JHDK were determined through RT-qPCR analysis. The expression of the MFE, JHEH, and JHDK genes was highly variable throughout the different developmental stages of the CBW (Figure 2). These genes were highly expressed primarily in larval and prepupal stages. Notably, the peaks in the expression of JHEH and JHDK were observed in the late third-instar larvae and early prepupal stages. The highest expression of MFE was also observed in the last larval stage; however, the MFE expression level remained similar throughout the third-instar larval stage, greatly decreased in the prepupa, and then gradually decreased from prepupa to pupa. JHDK and MFE expression was significantly higher in the carcass than in the gut (from 80% to 99% higher), whilst no significant difference in JHEH expression was observed between the carcass and gut (Supplementary Figure S4).

Overall, our results showed that gene silencing levels were dose dependent. The amount of naked dsRNA required to induce significant gene silencing was variable, depending on the target gene. All dsRNA doses tested led to a significant reduction in JHDK expression, while 0.5 or 2 µg of dsMFE repressed MFE expression (Figures 3A, B). However, only the dose of 2 µg of dsJHEH silenced the target gene (Figure 3C). Surprisingly, CS-TPP-dsRNA complexes induced the same level of gene silencing as the naked dsRNAs and, in some cases, impaired or reduced the level of gene silencing. On the other hand, PEI nanoparticles complexed with dsRNA at all tested doses improved JHDK and JHEH gene silencing compared with naked dsRNA. Although dsMFE + PEI did not lead to significant enhancement in the level of gene silencing when applied at 2 µg, when compared with naked dsRNA, dsMFE + PEI at low and moderate doses (0.05 and 0.5 µg, respectively) induced significantly greater gene silencing than the naked dsRNA at the same doses. Among the treatments involving PEI, the highest levels of MFE, JHDK, and JHEH silencing were achieved with moderate (0.5 µg) and high (2 µg) doses of dsRNA-PEI (Figure 3). Finally, persistent silencing of the MFE, JHDK, and JHEH genes was observed in the larvae injected with the respective naked dsRNAs or dsRNA-PEI at 0.5 µg. Significant differences in the expression of the target genes were detected between the treated larvae and control group up to 96 h after injection (Supplementary Figure S5).

As the RNAi response of the CBW was enhanced by PEI nanoparticles but not by CS-TPP, our subsequent experiments were performed only with PEI nanoparticles. In addition, as the dsRNA dose of 0.5 µg induced the same level of gene silencing as a 2 µg dose when the dsRNA + PEI complex was injected into the larvae, we selected the 0.5 µg dose for subsequent bioassays.

We further investigated the effects of delivering multiple dsRNAs complexed with PEI nanoparticles on target gene expression (Figure 4). Gene silencing efficiency was significantly higher when single dsRNAs were injected into the insects than when a combination of dsRNAs were injected. The silencing of JHEH or JHDK did not induce transcriptional changes in MFE (Figure 4A). However, JHDK silencing induced upregulation of JHEH and vice versa (Figures 4B, C). Furthermore, simultaneous silencing of JHDK and JHEH prevented the upregulation of either gene.

Regarding the effect of naked dsRNAs and PEI nanoparticles on CBW development and survival, we found that the dsJHEH/JHDK + PEI (18%), dsJHEH + PEI (42%), dsJHEH/JHDK (47%), and dsJHEH (75%) treatments induced significantly lower survival rates than the respective dsGFP + PEI (70%) and dsGFP (95%) controls (Figure 5). These results demonstrated that PEI nanoparticles and the combination of certain dsRNAs reduced CBW survival and affected its development (Figure 6). However, this result was not consistent across all target genes tested in this study. In fact, only the injection of third-instar larvae with dsJHEH/JHDK, naked or complexed with PEI, resulted in malformed adults. Malformed individuals showed an incomplete transition to the adult stage. When compared with normal adults, malformed insects had smaller elytra, which were completely tanned and hardened but positioned on the ventral side rather than on the dorsal side. This phenotype was not observed in any of the remaining treatments. However, most surviving individuals treated with dsRNA targeting JHEH (65%) or JHDK (54%) presented delayed adult development (Figure 6D).

We further investigated the possibility of using PEI nanoparticles as carriers to deliver dsRNA to the CBW through feeding. The combination of dsJHEH and dsJHDK, which induced the lowest survival rate when delivered by injection, was chosen for testing in feeding bioassays. The expression of JHDK was reduced significantly (55%) in larvae that were fed dsJHEH/JHDK + PEI at 1,000 ng/g compared with the dsGFP + PEI control. However, neither dsJHEH/JHDK nor dsJHEH/JHDK + PEI at 100 ng/g or dsJHEH/JHDK at 1,000 ng/g induced JHDK silencing (Figure 7A). On the other hand, CBW larvae that were fed dsJHEH/JHDK and dsJHEH/JHDK + PEI at 1,000 ng/g and dsJHEH/JHDK + PEI at 100 ng/g showed up to 56% JHEH silencing compared with the controls (Figure 7B).

Both low- and high-dose dsJHEH/JHDK + PEI significantly reduced the survival rates (56%–65%) of CBW compared with the controls (90%–93%). Furthermore, naked dsJHEH/JHDK at 1,000 ng/g resulted in slightly lower survival than that observed in the controls (Figure 8A). Surprisingly, larvae that were fed dsGFP + PEI did not exhibit significant mortality, as previously observed in the injection bioassays. In addition, we found that the larval stage duration of surviving insects that were fed dsJHEH/JHDK + PEI at 1,000 ng/g was significantly longer than that of the control group (Figure 8B). With respect to developmental alterations, the abnormal phenotypes observed in insects that were fed dsJHEH/JHDK + PEI were the same as those that were associated with simultaneous silencing of JHEH and JHDK induced by dsRNA injection (Figure 6).