RNA interference cannot be operated in lepidopteran insect? A nanocarrier breaks bottlenecks at all developmental stages of Spodoptera frugiperda

doi:10.21203/rs.3.rs-2127285/v1

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RNA interference cannot be operated in lepidopteran insect? A nanocarrier breaks bottlenecks at all developmental stages of Spodoptera frugiperda

https://doi.org/10.21203/rs.3.rs-2127285/v1

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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 1^st-2^nd 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.

Gene delivery

Nanocarrier

Nano-delivery system

Pest management

RNA interference

Nanocarrier (SPc)-loaded dsRNA can penetrate eggshell and cuticle of early instar larvae
SPc can protect dsRNA from degradation by midgut fluid and pupal hemolymph
SPc-based RNAi system is constructed for efficient RNAi at all developmental stages of fall armyworms
Our study provides a powerful tool for identifying functional genes in lepidopteran insects

Double-stranded RNA (dsRNA)-triggered RNA interference (RNAi) has been widely used in functional analysis of insect genes and pest management since its discovery in nematode Caenorhabditis elegans (Fire et al. 1998; Bellés 2010; Zhu and Palli 2020). In coleopteran insects, high-efficiency gene knockdown can be easily achieved toward various genes (Palli 2014). Whereas, lepidopteran insects are insensitive to dsRNA and the major limitation for successful RNAi includes: the strong dsRNA degradation by dsRNAases, accumulation of dsRNA in acidic granules and core RNAi machinery deficiency (Huvenne and Smagghe 2010; Wang et al. 2016; Yoon et al. 2017; Cooper et al. 2019). Therefore, a powerful and simple method is badly needed for dsRNA protection and efficient delivery, which can improve RNAi efficiency for the identification of functional genes in lepidopteran insects.

With the great development of nanotechnology in the past decade, a variety of nanoparticles such as chitosan, liposomes, cationic dendrimers, etc. have been designed and constructed for small interfering RNA (siRNA)/dsRNA delivery (Yan et al. 2021). The nanocarrier-mediated RNAi has been successfully achieved in Drosophila melanogaster (Whyard et al. 2009), Apis mellifera (Li-Byarlay et al. 2013), Anopheles gambiae (Zhang et al. 2015), Blattella germanica (Lin et al. 2017), Tribolium castaneum (Avila et al. 2018), Aphis glycines (Zheng et al. 2019; Yan et al. 2020) using various kinds of nanocarriers. In lepidopteran insects, Chitin synthase B of Spodoptera exigua can be nearly complete knockdown by guandine-containing polymer-loaded dsRNA through oral feeding method (Christiaens et al. 2018). In Chilo suppressalis, chitosan, carbon quantum dot and lipofectamine2000 were complexed with dsRNA to perform RNAi, and the target gene expression was significantly inhibited in all three groups (Wang et al. 2020). In most cases, nanoparticles can protect dsRNA from the degradation by endonuclease and help dsRNA escape from the endosomal accumulation (Gurusamy et al. 2020; Ma et al. 2022a). However, the research on nanocarrier-mediated RNAi is mostly through oral feeding method and focuses on single developmental stage of lepidopteran insects (Yan et al. 2021).

Our studying group has designed and constructed a facile-synthesized star polycation (SPc) for dsRNA and pesticide delivery (Li et al. 2019; Yan et al. 2019, 2022; Jiang et al. 2022; Ma et al. 2022b). A SPc-mediated transdermal dsRNA delivery system is established and has been applied to control Grapholita molesta via inhibiting feeding behavior (Yan et al. 2020; Wei et al. 2021). Subsequently, this delivery system is applied to disturb the wing development in Myzus persicae and Bactrocera dorsalis (Guo et al. 2021; Zhang et al. 2021). A recent publication reveals that this delivery system can be applied to co-deliver dsRNA and botanical pesticide to overcome the short life disadvantage of dsRNA and slow-acting property of botanical pesticide (Li et al. 2022). Whether this delivery system can be applied in lepidopteran insects for efficient and stable RNAi is an interesting topic.

Fall armyworm (Spodoptera frugiperda, Lepidoptera: Noctuidae) is a polyophagous pest damaging 353 plant species from 76 families, and it is well known for its robust migration ability (Westbrook et al. 2016; Montezano et al. 2018). In the current study, we established an efficient SPc-based RNAi system with soaking, topical application, oral feeding and injection methods that ensure efficient RNAi covering all developmental stages of fall armyworms (Fig. 1). For egg and early instar larvae, we tested the penetrability of SPc-loaded dsRNA across the eggshell and cuticle, and achieved efficient RNAi through soaking method and topical application, respectively. For mid-late instar larvae, we analyzed the stability of SPc-loaded dsRNA treated with midgut fluid, and achieved efficient RNAi through oral feeding method. For pupae and adults, we applied the injection method to deliver SPc-loaded dsRNA, and achieved efficient RNAi. When a hey developmental gene, SfCDA1, was silenced by SPc-mediated RNAi system, severe phenotypes of development inhibition and death were induced. The current study provides an efficient and simple method for identifying functional genes in lepidopteran insects, which is also beneficial for the development of RNAi-based pest management.

Insect rearing

S. frugiperda larvae, bought from Keyun Biological and Pesticide (China), were fed on artificial diet under 16:8 h light: dark photoperiod and 60–80% relative humidity at 25 ± 1°C. To avoid cannibalism, the 3rd instar larvae were reared separately until adult emergence. Adults were fed with 5% honey solution.

Gene Cloning And Dsrna Synthesis

Total RNA was extracted using RNA simple Total RNA Kit (Tiangen) and then prepared for cDNA synthesis using Prime-ScriptTM RT reagent Kit (TaKaRa, Japan). The V-type proton ATPase subunit d (SfATP-d) (GenBank accession No. XM_035590034.1) and SfCDA1 (GenBank accession No. XM_035590780.1) sequences were obtained from NCBI (http://www.ncbi.nlm.nih.gov). The primers were designed using NCBI primer 3 tools (Untergasser et al. 2012) and synthesized by Tsingke Biotechnology (China) (Table S1). The PCR thermal profile was: 1 cycle at 94°C for 3 min, followed by 32 cycles at 94°C for 30 s, 60°C for 30 s and 72°C for 45 s, with a final extension at 72°C for 5 min. The PCR products were cloned into pMD19T-Vector (TaKaRa) and further sequenced by Tsingke Biotechnology (China). The dsRNAs were prepared using T7 RiboMAXTM Express RNAi System (Promega, USA). The concentration and quantity of dsRNA were determined by NanoDrop 2000 spectrophotometer. The dsRNA of enhanced green fluorescent protein (eGFP) was used as control in all RNAi experiments.

Stability Analysis Of Reference Genes For Quantitative Real-time Pcr

To investigate the stability of reference genes in different tissues and developmental stages, more than 300 eggs, 20 first-instar larvae, 10 second-instar larvae, 10 third-instar larvae, 5 fourth-instar larvae, 5 fifth-instar larvae, 5 sixth-instar larvae, 5 pupae and 5 adults were collected separately to prepare the cDNA samples, and each treatment contained independent 5 samples. Seven genes, acetylcholinesterase (ACE), Actin-5C (Actin), beta tubulin (β-TUB), elongation factor 1-gamma (EF-γ), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S18 (RPS18) and ribosomal protein L27 (RPL27), were selected as the candidate reference genes. The primer for GAPDH (GenBank accession No. OK319027.1) was designed using NCBI primer 3 tools, and primers for other candidate genes were described by Zhou et al. (2021). The cycle threshold (Ct) of each gene was obtained by quantitative Real-Time PCR (qRT-PCR) using TranStart® Top Green qPCR SuperMix (+ Dye II). The qRT-PCR was performed on QuantStudio 6 Flex platform, and its conditions were set as follows: 95°C for 5 min, and 40 cycles of 95°C for 15 s, 57°C for 30 s and 72°C for 30 s. The geNorm, NormFinder and bestkeeper software were used to evaluate the expression stability of candidate reference genes.

Spatiotemporal expression analysis of SfATP-d and SfCDA1.

Fall armyworms were collected at all developmental stages from eggs to adults, and various tissues such as head, Malpighian tubule, epidermis, fat body, midgut and testis were also collected for cDNA preparation. The Ct value was determined using qRT-PCR. Standard curves were established for SfATP-d, SfCDA1 and GAPDH with five serial dilutions (3ⁿ-fold) (Fig. S1). The qRT-PCR correlation coefficient (R²) and amplification efficiency (E) was determined (Radonić et al. 2004).

Infiltration Assay Of Spc-mediated Dsrna Delivery System In Eggs And 2nd Instar Larvae

The Fluorescein RNA Labeling Mix (Roche, Switzerland) was used to label dseGFP. The SPc was mixed with dsRNA at the mass ratio of 1:1 for 15 min at room temperature according to the method described by Ma et al. (2022a). The final concentration of 1% alkyl glucoside (APG) was then added to decrease the surface tension of droplet. The newly-laid eggs were collected, and 50 µL fluorescent dseGFP/SPc complex was added for 5 min. The 0.5 µL fluorescent dseGFP/SPc complex was applied on the notum of 2nd instar larvae using a microinjector (World Precision Instruments, Germany). SPc and naked dseGFP were used as controls. The treated eggs and 2nd instar larvae were washed by ddH₂O, and photos were captured using EVOS FL fluorescence microscope (AMG, USA). The fluorescence intensity was calculated using ImageJ 1.8 software (National Institutes of Health, USA). Each treatment contained 10 eggs or larvae, which was repeated 3 times.

Stability Test Of Spc-loaded Dsrna Treated With Midgut Fluid And Pupal Hemolymph

To obtain the midgut fluid, the 6th instar larvae were stabilized and squeezed, and the exhaled midgut fluid was gathered. Pupae were scissored along the abdomen, and the hemolymph was aspirated with a pipette. Each sample was collected from at least 5 larvae or pupae, and N-phenylthiourea was then added at the final concentration of 1 µg/µL to inhibit melanism.

To test the stability of dsRNA, midgut fluid and pupal hemolymph were diluted with 1×PBS, and various concentrations of midgut fluid or pupal hemolymph were incubated with 1 µg dseGFP at 25°C for 4 h. The integrity of dsRNA was detected by agarose gel electrophoresis. To investigate the stability of SPc-loaded dsRNA, the dseGFP (1 µg) was mixed with SPc to obtain dseGFP/SPc complex, which was incubated with the midgut fluid (1%) or pupal hemolymph (6.25%) at 25°C for 4 h. Then the complex was decomposed in 0.1% SDS solution and further analyzed. The ImageJ 1.8 software was used to determine the relative band intensity, and each treatment was repeated 3 times.

Establishment Of Rnai Methods For Fall Armyworms

Soaking method for egg RNAi. The 200 µL of dsATP-d (50/100/300 ng/µL)/SPc/APG complex was incubated with eggs for 1/3/5 min. The absorbent paper was then used to remove the excessive complex formulation. The eggs were further treated with liquid nitrogen at 24/48/72 h after the soaking and used to synthesize cDNA. To determine the potential negative effects of soaking method, the eggs were soaked in various formulations for 3/5/7 min, and the hatching rate was finally recorded. Each treatment contained 20 newly-laid eggs and was repeated 5 times.

Topical application for early instar larvae RNAi. The 0.5 µL of dsATP-d (0.1/0.5/1 µg)/SPc/APG complex was applied on the notum of each 2nd instar lava. The topical application was repeated twice and thrice at 24 h after the first and second application to increase the RNAi efficiency. At 24/48/72 h after the first application, larvae were collected, treated with liquid nitrogen, and used to synthesize cDNA.

Oral feeding method for mid-late instar larvae RNAi. The 4th and 6th instar larvae were firstly starved for 12 h, and then fed on artificial diet containing dsATP-d (100/150/200 ng)/SPc complex and dsATP-d (1/3/5 µg)/SPc complex, respectively. The oral feeding method was also repeated twice and thrice at 24 h after the first and second application. The remaining food was removed after 12 h feeding, and the larvae were starved for another 12 h before the next application. At 24/48/72 h after the first application, larvae were also prepared for cDNA synthesis.

Injection method for pupa and adult RNAi. Adults were pre-anesthetized with CO₂ before injection, and pupae and adults were injected with 5 µL formulation of dsATP-d (5/7.5/10 µg)/SPc complex at the intersegmental membrane in the 8th -9th abdomen. At 24/48/72 h after the injection, samples were collected for cDNA synthesis. The emergency rate of pupae and survival rate of adults were finally recorded. Each treatment contained 20 pupae/adult and was repeated 3 times.

The above cDNA samples were used to examine the RNAi efficiencies of target genes. The dseGFP was applied as control. The GAPDH was chosen as reference gene and the amount of SfATP-d expression was normalized using the 2^−ΔΔCt method (Livak and Schmittgen 2001). Each treatment contained 3 independent samples.

RNAi effects of SfCDA1 gene through SPc-mediated RNAi system

The SfCDA1 gene was taken as an example to test the SPc-mediated RNAi system. The formulation of dsCDA1 (1 µg)/SPc/APG complex was applied on the notum of 1st instar 3rd day larvae for 4 days, and the 3rd instar larvae were fed with dsCDA1 (2 µg)/SPc/APG complex for 3 days. The relative expression of SfCDA1 was detected on 4 d after RNAi. The dseGFP/SPc complex was used as control. The mortality, body length and body weight were recorded on the first day of each instar. Each treatment contained 20 larvae and was repeated 3 times.

Statistical analysis

The statistical analyses were conducted using SPSS 20.0 software (SPSS Inc. USA). The data were analyzed using the ANOVA with Tukey HSD test or independent t-test at the P = 0.05 level of significance, and the descriptive statistics are shown as the mean value and standard deviation of the mean.

Reference gene screening and RNAi target gene selection

The expression stabilities of seven commonly used reference genes were evaluated by geNorm, NormFinder and BestKeeper. The current data showed that GAPDH and β-tubulin were the most stable and unstable reference genes, respectively (Table 1). The complete ranking and specific M and SD values are shown in Table S2. Thus, the GAPDH was used as reference gene in following experiments.

Table 1

Comprehensive ranking of candidate reference genes.
Reference gene	Egg	2nd larva	4th larva	6th larva	Pupa	Adult	Average
GAPDH	2.7	3.7	2	5	2.3	3	3.1
RPL27 Actin EF-γ ACE RPS18 β-tubulin	3.3 3.7 3.3 3 5 7	5 2.3 2.7 4 3.7 6.7	2 6 2 4.3 7 4.6	6.7 2.7 6.3 2 2.3 3	1.7 5.3 4 5.7 2 7	2 1 4.3 4.7 7 6	3.4 3.5 3.8 3.9 4.5 5.7

As shown in Fig. 2A, we selected ATP-d gene and synthesized the dsRNA fragment in its conserved domain. Among tested six tissues, the lowest Ct value (21.6) was observed in the midgut, and the highest value was 24.5 in the head (Fig. 2B). Meanwhile, the Ct value fluctuated between 20.6 and 24.9 during the life cycle after a rapid reduction in the egg stage (Fig. 2C). These results revealed that the expression of SfATP-d was high and stable in various tissues and developmental stages. Therefore, the SfATP-d was suitable as target gene for the establishment of SPc-based RNAi system.

Spc-based Soaking Method For Egg Stage

The soaking method was chosen for convenient RNAi in egg stage, and the permeability of SPc-loaded dsRNA was firstly tested. The eggs treated with fluorescent dseGFP/SPc complex exhibited the strongest green fluorescence (Fig. 3A), and its relative fluorescence intensity was more than 6.3-fold compared to the eggs treated with dseGFP alone (Fig. 3B).

To test the RNAi efficiency of various operation methods, a consistently expressed gene SfATP-d, which seems to be nonvital for development, was selected. As shown in Fig. 3C, soaking with 300 ng dsATP-d alone could not down-regulate SfATP-d expression (Fig. 3C). In contrast, the expression of SfATP-d could be significantly inhibited by SPc-delivered dsATP-d at much lower application dose (Fig. 3D). To determine the RNAi persistence, 3 time points were selected to test the RNAi efficiency, and the mRNA level of target gene was still lower than that of control group at 72 h after soaking (Fig. 3E). Meanwhile, soaking method (≤ 5 min) did not influence the egg hatching (Fig. S2), and the RNAi efficiency increased with longer soaking duration (Fig. 3F).

Spc-based Topical Application For Early Larval Stage

The topical application was chosen for convenient RNAi in early instar larvae. As shown in Fig. 4A, the fluorescent dseGFP/SPc complex could penetrate the cuticle of 2nd instar larvae, and its relative fluorescence density was 3.8-fold and 11.6-fold to those in dseGFP and SPc treatment, respectively (Fig. 4B). As shown in Fig. 4C and 4D, the RNAi efficiency was remarkably improved by adding SPc, and higher amount of dsATP-d could improve RNAi efficiency in a dose dependent manner. However, the gene expression was rapidly reverted to normal level at 72 h after the topical application (Fig. 4E). Therefore, repeated RNAi operation was needed for persistent RNAi (Fig. 4F).

Spc-based Oral Feeding Method For Mid-late Larval Stage

As shown in Fig. S3, the topical application was no longer suitable for RNAi in 3rd instar larvae. Thus, the oral feeding method was applied for RNAi in mid-late instar larvae. Based on current study, the midgut fluid of 6th instar larvae could degrade dseGFP completely within 4 h (Fig. 5A). Interestingly, SPc could protect the dseGFP from degradation by midgut fluid (Fig. 5B), and the relative band intensity of SPc-loaded dseGFP showed no significant change after midgut fluid treatment (Fig. 5C).

As expected, dsATP-d alone could not suppress gene expression in 4th and 6th instar larvae through oral feeding method (Fig. 5D and 5H). Whereas, the SfATP-d expression of 4th instar larvae could be decreased by 70.8 ± 8.3% through oral feeding of SPc-loaded dsATP-d (0.2 µg) (Fig. 5E). As the growth of larvae, 3 µg dsATP-d/SPc complex was sufficient for gene knockdown in 6th instar larvae (Fig. 5I). Similarly, the gene expression tended to revert to normal level within 72 h for both 4th and 6th instar larvae (Fig. 5F and 5J), and the recovery of gene expression could be solved by repeated RNAi operations (Fig. 5G and 5K).

Spc-based Injection Method For Pupal And Adult Stages

Similar to the strong stability of SPc-loaded dsRNA treated with midgut fluid, SPc could also prevent dseGFP degradation by 6.25% pupal hemolymph within 4 h (Fig. 6A-C). Since the injection could cause mechanical damage, we firstly tested the emergence rate of pupae and survival rate of adults after injection. As shown in Fig. S4, injection of 5 µL formulation did not induce any negative effects toward pupae and adults. As expected, 10 µg dsATP-d alone could not silence the target gene expression in pupae and adults (Fig. 6D and 6G). With the help of SPc, the gene expression could be successfully inhibited (Fig. 6E and 6H), and the RNAi effect was persistent within 72 h (Fig. 6F and 6I).

Application of SPc-mediated RNAi system toward SfCDA1

To test the potential of above methods for pest control, a key developmental gene SfCDA1 was selected. It contains an open reading frame (ORF) of 1629 bp encoding 542 amino acids, which includes the chitin-binding peritrophin-A domain (CBD), low-density lipoprotein receptor class A domain (LDLa) and CDA catalytic domain (Fig. 7A). Spatiotemporal expression analysis revealed that SfCDA1 was highly expressed in epidermis and 2nd instar 1st day larvae.

Based on the expression pattern of SfCDA1, the 1st instar 3rd day larvae were treated with SPc-loaded dsCDA1 though topical application for 4 d, and oral feeding method was then applied to 3rd instar larvae for 3 d. After 4 d of RNAi, the SfCDA1 expression was significantly decreased by 66% compared to the control group (Fig. 7D). The persistent knockdown of SfCDA1 caused 40% mortality in larval stage (Fig. 7E). Furthermore, the body length and weight of survival larvae were also significantly reduced in different degrees (Fig. 7F-G), and the growth of survival larvae was severely inhibited (Fig. 7H).

RNAi has been widely applied to analyze insects’ gene function since its first application in identifying frizzled and frizzled 2 in D. melanogaster (Kennerdell and Carthew 1998; Bellés 2010). Compared to newly-emerging technology gene editing, siRNA/dsRNA-triggered RNAi has the advantages of simple operation, low cost and short time for functional gene analysis (Boettcher and McManus 2015). However, the quick degradation of siRNA/dsRNA by nucleases and low delivery efficiency restrict the application of RNAi technology in insensitive insects (Wang et al. 2016; Peng et al. 2020). Various nano-delivery systems using liposomes, polymers and peptides, etc. were constructed to enhance the RNAi efficiency (Zhang et al. 2010; Hunter and Sinisterra-Hunter 2018; Christiaens et al. 2020; Gurusamy et al. 2020). Constructing an efficient nanocarrier-based RNAi system for whole developmental stages of insensitive lepidopteran insects is a big challenge in the past decade.

In the current study, we explored to construct a convenient RNAi system for whole developmental stages of fall armyworms. For qRT-PCR analysis, the suitable target gene and reference gene with stable expression level and no lethal effect should be firstly selected to construct the proper studying system. The GAPDH gene was used as the reference gene due to its stable expression at various development stages of fall armyworms. The vacuolar (H⁺)-ATPase (V-ATPase) is a proton pump with various subunits, such as A, B, C, D, E, F, G, H, a, c, c’, c’’ and d, which is highly conserved in eukaryotic cells (Nishi and Forgac 2002; Wieczorek et al. 2009; Li and Xia 2012). Our result revealed that the expression level of SfATP-d gene was relatively stable among various tissues and developmental stages. Furthermore, the RNAi of SfATP-d gene did not influence the S. frugiperda development. Thus, the SfATP-d gene was suitable and used for the establishment of nanocarrier-based RNAi system.

Soaking method has been used to trigger RNAi responses in nematodes and insect cell lines for its convenient operation (Tabara et al. 1998; Yu et al. 2013; Clemens et al. 2000). However, very limited RNAi efficiency is observed by soaking Helicoverpa zea eggs in dsRNA solution. (Wang et al. 2018). Our results showed that the SPc-loaded dsRNA could penetrate the eggshell and cuticle quickly, and the efficient RNAi was achieved in eggs and early instar larvae through SPc-based soaking method and topical application. For egg RNAi, soaking with more than 100 ng/µL dsRNA for less than 5 min could effectively silence the target gene expression. For early instar larva RNAi, topical application of more than 0.5 µg dsRNA could significantly down-regulate the target gene expression. However, the gene expression could rapidly revert to normal level at 72 h after the topical application in early instar larvae, and topical application could be repeated every 24 h to achieve persistent gene knockdown. The current study expanded the application area of SPc-mediated transdermal dsRNA delivery system to insensitive lepidopteran insects.

The topical application could not achieve high RNAi efficiency in mid-late instar larvae of fall armyworms, thus we explored to apply oral feeding method to establish an efficient RNAi system. However, the rapid degradation of dsRNA by dsRNAases and its accumulation in endosomes are two main reasons that constrain the successful RNAi in lepidopteran insects (Wang et al. 2016; Yoon et al. 2017). Our previous study has demonstrated that SPc can prevent dsRNA from degradation by RNase A and hemolymph, and SPc-delivered dsRNA exhibits reduced accumulation in endosomes of Sf9 cells (Ma et al. 2022a). Herein, the dsRNA protection by SPc was tested with midgut fluid, and much stronger stability of SPc-loaded dsRNA was observed in the current study. The qRT-PCR results also supported the successful RNAi through SPc-based oral feeding method toward mid-late instar larvae. We recommended to feed 4th and 6th instar larvae with 100 ng and 3 µg dsRNA for efficient RNAi, respectively. In addition, the midgut fluid of starving larvae exhibits the lower dsRNA-degrading activity (Rodríguez-Cabrera et al. 2010). Therefore, the starvation treatment in our experiment might be another important factor for efficient gene knockdown.

Injection method is suitable for pupal and adult RNAi because it can avoid the structural barriers and deliver dsRNA directly into insect body (Zhang et al. 2018). Similarly, the SPc could protect the dsRNA from pupal hemolymph degradation, and the target gene expression could be significantly inhibited in pupae by SPc-loaded dsATP-d (10 µg). For adult RNAi, we recommended to apply 10 µg dsRNA for efficient gene knockdown.

CDAs that deacetylate chitin to form chitosan belong to the carbohydrate esterase family 4 (CE-4) of the carbohydrate active enzymes (CAZY), which was taken as an example to illustrate the application of SPc-based RNAi system (Xi et al. 2015). In D. melanogaster, the function of DmCDA1 is correlated to tracheal tube development and morphology (Luschnig et al. 2006; Wang et al. 2006). In Leptinotarsa decemlineata, silencing chitin deacetylase 2 can affect the chitin accumulation and lead to approximately 20% mortality of larvae (Wu et al. 2019). Due to the high expression of SfCDA1 gene in epidermis and 2nd instar 1st day larvae, we applied the topical application and oral feeding method for seven days. Compared to control group, SfCDA1 could be successfully knocked down, and the growth and development of larvae were severely influenced. The above results suggested that our SPc-mediated RNAi system had great potential in lepidopteran pest management. Moreover, Li et al. (2022) has demonstrated that the control efficacy can be improved by co-delivering dsRNA and pesticides, and co-delivery of dsCDA1 and lufenuron may achieve a desired control efficacy for fall armyworms (Zhang et al. 2022).

Ethical approval

Not applicable.

Competing interests

The authors declare that they have no conflict of interest.

Authors’ contributions

JS, SY and ZC conceived and designed research. ZC, ZM and YZ conducted experiments. ZC and SY analyzed data. ZC, SY and JS wrote the manuscript. All authors read and approved the manuscript.

Funding

This research was supported by the National Key R&D Program of China (2021YFC2600404) and National Natural Science Foundation (32030012).

Availability of data and materials

Not applicable.

Supplementary information

The online version contains supplementary material available at XXXXXX.

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RNA interference cannot be operated in lepidopteran insect? A nanocarrier breaks bottlenecks at all developmental stages of Spodoptera frugiperda

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Abstract

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Key Message

Introduction

Materials And Methods

Insect rearing

Gene Cloning And Dsrna Synthesis

Stability Analysis Of Reference Genes For Quantitative Real-time Pcr

Infiltration Assay Of Spc-mediated Dsrna Delivery System In Eggs And 2nd Instar Larvae

Stability Test Of Spc-loaded Dsrna Treated With Midgut Fluid And Pupal Hemolymph

Establishment Of Rnai Methods For Fall Armyworms

Statistical analysis

Results

Spc-based Soaking Method For Egg Stage

Spc-based Topical Application For Early Larval Stage

Spc-based Oral Feeding Method For Mid-late Larval Stage

Spc-based Injection Method For Pupal And Adult Stages

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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