Characterization and potential mechanism of resistance to double-stranded RNA in willow leaf beetle, Plagiodera versicolora

doi:10.21203/rs.3.rs-3250534/v1

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Characterization and potential mechanism of resistance to double-stranded RNA in willow leaf beetle, Plagiodera versicolora

https://doi.org/10.21203/rs.3.rs-3250534/v1

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published 24 Apr, 2024

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RNAi-based pesticides have emerged rapidly in recent decades and are believed to be the third generation of pesticides. Although two case studies of the resistance to RNA pesticides have been reported in western corn rootworm and Colorado potato beetle, whether RNAi-resistance is general phenomena in other coleopteran insects and the underlying mechanism of resistance to RNA pesticides are largely unknown. Here we report the development of a highly (> 4110-fold) dsRNA-resistant population (Pv-30R) of Plagiodera versicolora by feeding a laboratory-rearing susceptible population (Pv-S) with the leaves of willow plants after seven episodes of selection using foliar coating dsRNA targeting a signal recognition particle protein 54k gene. We showed that Pv-30R was cross-resistant to another dsRNAs (dsActin and dsSnap) but susceptible to the Cry3Bb protein from Bacillus thuringiensis, and the resistance was an autosomal and recessive trait. Although no significant differences of the dsRNA stability in the midgut of larvae between Pv-S and Pv-30R were observed, uptake of dsRNA in the midgut tissue of larvae from Pv-30R was impaired. Overall, these results demonstrate that high-levels of resistance to RNA pesticides can developed quickly in P. versicolora in laboratory condition. These findings highlight the requirements to counter the potential rapid evolution of insect resistance to dsRNA in the field.

RNAi

dsRNA resistance

cross-resistance

resistance mechanism

Plagiodera versicolora

RNAi-based pesticides have emerged rapidly in recent decades. To assure the sustainability for RNA pesticides utilization, the potential evolution of resistance to RNA pesticides in pests need to be taken into account.

A highly dsRNA-resistant strain of a forest pest was obtained and the characteristics of resistance to dsRNA were systematically investigated, after seven episodes of continuous selection with increasing concentration of insecticidal dsRNA.

Our findings highlight the requirements to counter the potential rapid evolution of insect resistance to dsRNA in the field.

RNA interference (RNAi) is a highly conserved mechanism induced by double-stranded RNA (dsRNA) to trigger the degradation of endogenous cellular mRNA (Fire et al. 1998). RNAi has been found in all eukaryotic organisms, including invertebrates, vertebrates, fungi, plants and algae, and is involved in function as an antiviral innate immune response (Agrawal et al. 2003; Cullen 2014). Due to its highly-specific function in gene silencing, RNAi has been widely used as a powerful reverse genetic tool for gene function study and recently also shown its great potential in pest management (Baum et al. 2007; Nandety et al. 2015; Price and Gatehouse 2008; Suzuki et al. 2017). RNA pesticides have emerged as various products including spraying dsRNA formulation, nanoparticle-coated dsRNA, and dsRNA-expressed and insect-resistant transgenic plants (Niu et al. 2022; Rodrigues et al. 2021; Zhang et al. 2015). RNAi pesticides are environment-friendly, highly-specific to target pests, harmless to humans and animals (Fletcher et al. 2020). RNAi pesticides is therefore promising to become the third generation of pesticides following chemical pesticides and Bacillus thuringiensis (Bt) toxins (Fletcher et al. 2020; Ni et al. 2017).

Like that occurred for chemical pesticides and Bt toxins, it is not surprise that insect could also develop the resistance to RNA pesticides (Gassmann et al. 2009). Up to now, there are two case studies of resistance to RNA pesticides in different coleopteran pest insects (Khajuria et al. 2018; Mishra et al. 2021). Data from both cases suggested that, resistance to RNA pesticides can develop rapidly to a high-level of resistance after less than 10 episodes of selection, as though different delivery methods of RNA pesticides were used for the selection (Khajuria et al. 2018; Mishra et al. 2021). In addition to that, the dsRNA-resistant strains in both cases were shown to be cross-resistant to alternate dsRNA other than the insecticidal dsRNA used for selection. Similar with the common resistance to chemical insecticides and Bt toxins, both reported resistance to dsRNA were recessive and autosomal inherited. Understanding the knowledge of mechanism of resistance can provide guidance for strategy designing to delay the rapid evolution of resistance in the field (Jurat-Fuentes et al. 2021). However, little is known about the development process and mechanism of resistance to dsRNA.

To fill the gap of characteristics and mechanism of resistance to dsRNA in forest pest insect, here we chose the willow leaf beetle, Plagiodera versicolora, which feeding the leaf of plants in Salicaceae family, to select dsRNA-resistant trait with foliar delivery of dsRNA. After seven episodes of continuous selection with increasing concentration of insecticidal dsRNA, a highly dsRNA-resistant strain of P. versicolora was obtained and the characteristics of resistance to dsRNA were systematically investigated.

2.1 Insect rearing

The P. versicolora adults were originally collected from Sha Lake Park in Wuhan city, Hubei province, China (30°28′47″N, 114°21′33″E). Insects were reared with fresh willow leaves in growth chambers at 28 ± 0.5°C under a 16L: 8D photoperiod and 60 ± 5% relative humidity (RH). The willow leaves used for insect rearing were collected from Salix babylonica L grown in greenhouse. The susceptible population of P. versicolora (Pv-S) was reared in laboratory for multiple generations without exposure to chemical insecticides.

2.2 In vitro dsRNA synthesis

The gene sequences of Srp54k, Actin and Snap of P. versicolora were obtained from transcriptome sequencing (Majorbio, China). Fragments of GFP, Srp54k, Actin and Snap were amplified using gene-specific primer pairs containing the T7 promoter sequence (Table S1) and purified with a gel extraction kit (Omega, China). In vitro dsRNA synthesis was performed using the T7 RiboMAX™ Express RNAi System (Promega, USA) according to the manufacturer’s instruction. The yield of RNA was determined by ultraviolet absorbance at a wavelength of 260 nm using a Nano Photometer (IMPLEN, Germany).

2.3 Selection for resistance to RNA pesticides

The susceptibility of Pv-S to dsSrp54k was tested by foliar-overlaid bioassay to determine the median lethal concentration (LC₅₀) and 95% lethal concentration (LC₉₅) values of dsSrp54k. Briefly, eight concentrations of dsSrp54k diluted with 0.1% Tween-20 were initially prepared, including 0.05, 0.1, 0.25, 0.5, 0.75, 1.0, 1.5 and 2.0 ng/µL. And 0.1% Tween-20 without dsRNA was used as the blank control. Then, the young willow leaves were detached and soaked in 3 mL of the dsSrp54k solutions for approximately 10 seconds, and allowed to dry completely on a plastic wrap. Finally, the second instar larvae (n = 10) of P. versicolora were allowed to feed on the dsSrp54k-coated willow leaves. Four biological replicates were performed for each treatment. The survival rates of insects were recorded daily. Probit analysis was conducted using SPSS software to determine the LC₅₀ and LC₉₅ values of dsSrp54k..

For resistance selection, the concentration of dsSrp54k for the first round selection was determined as 0.27 ng/µL ( LC₉₅ value of dsSrp54k against the Pv-S strain). For the first round selection, a total of 800 second-instar larvae from the Pv-S strain were treated and those survivors were named as Pv-0.27R colony until the F1 progeny generated. For the second round selection, 80 F1 second instar larvae generated by Pv-0.27R adult were also treated with 0.27 ng/µL of dsSrp54k and the survival rated was determined as 53.75%. Then survivors from each round selection were treated with increasing concentration of dsSrp54k (Table 1). For comparison, the larvae from Pv-S were treated with the same concentration of dsSrp54k in each round selection and the survival rates of Pv-S after dsRNA exposure were recorded. Finally, the concentration of dsSrp54k was increased up to 30 ng/µL at the seventh round selection and the survival rate of Pv-10R was calculated as 88.9%, while larvae from Pv-S were all killed upon this treatment. Survivors from the seventh round selection were named as Pv-30R and the Pv-30R colony was reared with willow leaves coated with 30 ng/µL of dsSrp54k.

Table 1

Survival rates for each generation of Pv-S and Pv-R during the selection process of resistance to dsSrp54k in *P. versicolora*.
Generation of Pv-R	Colony	N^b	Concentration (ng/µL)	Survival rate (%)
1^a	Pv-S	800	0.27	4.37
1^a	Pv-0.27R	80	0.27	53.75
2	Pv-S	40	0.54	2.5
2	Pv-0.27R	40	0.54	67.5
3	Pv-S	40	1.08	0
3	Pv-0.54R	40	1.08	75
4	Pv-S	30	2	0
4	Pv-1.08R	30	2	60
5	Pv-S	20	5	0
5	Pv-2R	20	5	80
6	Pv-S	40	10	2
6	Pv-5R	40	10	86
7	Pv-S	45	30	0
7	Pv-10R	45	30	88.9
^a The first generation of Pv-R was named as Pv-0.27R, which was composed of survivors from the first round selection of Pv-S with 0.27 ng/µL (LC₉₅ value for Pv-S) of dsSrp54k. The survival rate for Pv-0.27R under 0.27 ng/µL of dsSrp54k was tested using the second-instar larvae generated from the Pv-0.27R adult survivors. Since the second generation, the concentration of dsSrp54k was gradually increased and the adult survivors under each concentration were renamed according to the value of tested concentration.
^b Number of tested larvae for each generation.

The relative level of resistance to dsSrp54k was further evaluated by foliar coated bioassays with higher concentration of dsRNA (300 ng/µL), and survival rates, leaf consumed area, mean weight of larvae and mRNA expression levels of Srp54k in Pv-S and Pv-30R were monitored.

2.4 Evaluating for crossresistance to other dsRNAs and Bt Cry3Bb protein

Based on the bioassay results in our previous study, β-Actin and Snap were proved to be two another effective targets for RNAi in P. versicolora (Xu et al. 2021). DsRNAs targeting β-Actin and Snap were synthesized in vitro and the survival rates of Pv-S and Pv-30R under exposure to dsActin and dsSnap were recorded daily. Consistent with the concentration of dsSrp54k, 30 ng/µL was chosen as the discriminatory concentration of dsActin and dsSnap, under which Pv-S were all dead at the 6th day post feeding. The larvae were collected individually after 4 days of feeding and stored at -80℃ until RNA extraction for gene expression analysis.

Cross-resistance to Bt Cry3 protein was tested with the leaves of transplastomic poplar plants expressing Cry3Bb protein (Pa-Cry3Bb), which has been shown toxic to the Pv-S (Xu et al. 2020). The wild-type poplar (Pa-wt) leaves were used as control. The survival rates of Pv-S and Pv-30R upon feeding on Pa-wt or Pa-Cry3Bb were recorded daily and the leaf damaged area was photographed and quantified after 4 days of feeding.

2.5 Evaluating inheritance of resistance to dsRNA

Reciprocal crosses of Pv-S and Pv-30R were performed to determine the inheritance of resistance to dsRNA in Pv-30R. The female and male adults emerged at the same day were collected simultaneously. Then, 10 adults from Pv-S and 10 adults from Pv-30R were reciprocally paired in the rearing box. The second-instar larvae of progenies from reciprocal crosses were fed with the willow leaves coated with 30 ng/µL of dsSrp54k, and their survival rates were recorded daily. For comparison, the survival rates of the second-instar larvae from Pv-S and Pv-30R were estimated under treatment with 30 ng/µL of dsSrp54k. The dominance value (h) was calculated according to the method reported previously (Liu and Tabashnik 1997).

2.6 RNA extraction, cDNA synthesis and qRT-PCR analysis

The total RNAs were isolated from insect samples using RNAiso Plus Reagent (Takara, Dalian, China) according to the manufacturer’s instruction. Hifair® II 1st Strand cDNA Synthesis Kit with gDNA digester (Yeasen, China) was used to synthesize cDNA. The relative expression levels of targeted genes were quantified by qRT-PCR with SYBR® Premix Ex Taq™ II (Tli RNase H Plus) (Takara, Dalian, China) in a Bio-Rad CFX Connect Real-Time System (Bio-Rad, USA). The reaction was performed in a final volume of 10 µL containing 5 µL of 2×SYBR® Premix Ex Taq™ II, 2 µL of cDNA and 0.25 µL of each primer (10 µM; Table S1). The reaction conditions were as follow: 95℃ for 2 min, followed by 40 cycles at 95℃ for 5 s, and then 60℃ for 30 s. The 40S ribosomal protein S18 (RPS18) gene was used as internal control. Each experiment was conducted with three biological replicates. Quantitative analysis of gene expression was calculated using the 2^−∆∆Ct method (Schmittgen and Livak 2008).

2.7 Investigation of degradation and uptake of dsRNA in the larvae midgut.

To investigate the degradation of dsRNA in the larvae midgut between Pv-S and Pv-30R, the gut juice of third-instar larvae from both Pv-S and Pv-30R was collected as described previously (Xu et al. 2021). Briefly, whole guts from 10 third-instar larvae were dissected, crushed in 100 µL ice-cold Ringer’s solution in a centrifuge tube, and then centrifugated at 12,000 g for 10 min for collection of the supernatants. The total protein concentrations of the fluid samples were determined using the BCA Protein Assay kit following the manufacturer’s instruction. The amount of the gut juice was normalized and adjusted to 1.0 µg/µL of total protein for both Pv-S and Pv-30R. 150 ng of dsSrp54k (dissolved in 5 µL nuclease-free water) were added to 5 µL of the gut juice, and incubated at 28°C for indicated times. DsSrp54k incubation with the same volume of Ringer’s solution was served as control. Subsequently, 2 µL of 6×loading dye (20% glycerol, 1.25 mM Na₂EDTA, 0.1% bromophenol blue, 0.1% xylene cyanol) was mixed with the 10 µL of samples, and analyzed by electrophoreses in 1% agarose gels.

To investigate the uptake of dsRNA by midgut cell in Pv-S and Pv-30R, fluorescein-labeled dsRNA was in vitro synthesized using Fluorescein RNA Labeling Mix (Roche, USA) and incubated with midguts dissected from the third-instar larvae under aseptic condition at 25℃ in the darkness for 16 h. After incubation, images of midguts were captured using 488 nm (fluorescein) and 360 nm (DAPI) laser for excitation and 517 nm (fluorescein) and 450–460 nm (DAPI) for emission filter sets, under a laser-scanning confocal microscope (LSM 980, Carl Zeiss, Germany).

2.8 Statistical analysis

Survival curves were analyzed using the Kaplan–Meier method, and the log-rank test was used to evaluate the significance of differences between two groups. One-way ANOVA followed by Tukey’s HSD multiple comparison test was used to compare the mean weight and relative expression levels of targeted gene among multiple groups. A value of P < 0.05 was considered significantly different. All data were statistically analyzed using SPSS version 19.0 and figures were drawn using GraphPad Prism 8.

3.1 Selection for resistance to dsRNA in P. versicolora

Bioassays with dsSrp54k showed the concentration of dsSrp54k killing 95% of the individuals (LC₉₅) was 0.275 (0.198–0.488) ng/µL, which was used as the initial selection concentration (Table 1, Fig. 1). For the first round of selection, a total of 800 of second-instar larvae from Pv-S were fed with the willow leaves coated with 0.27 ng/µL of dsSrp54k. The survival rate of P. versicolora after the first round exposure of dsSrp54k was 4.37% (Table 1), reflecting the reliability of LC₉₅ for Pv-S population. For the second round selection, a total of 80 F1 larvae generated by adult survivors from the first round selection were tested with the same concentration of dsSrp54k (0.27 ng/µL), and the survival rate was increased up to 53.75% (Table 1). Since the second generation, the survival rates for each generation under increasing dsSrp54k exposure from 0.54 ng/µL to 10 ng/µL were stable and all higher than 50% (Table 1), demonstrating the rapid evolution of resistance to dsSrp54k in Pv-R colony. After continuous seven episodes of selection with gradually increased concentration of dsSrp54k, the Pv-R colony exhibited resistant to a high concentration (30 ng/µL) of dsSrp54k. The survival rate for the seventh generation was determined as 88.9% when the larvae fed on the willow leaves coated with 30 ng/µL of dsSrp54k, while the survival rate of Pv-S was 2% (Table 1), indicating the activity of dsSrp54k and high-levels resistance in Pv-R colony. The resistant colony of P. versicolora (Pv-R) to 30 ng/µL of dsSrp54k was named as Pv-30R and maintained under exposure with 30 ng/µL of dsSrp54k.

Bioassays were performed to Pv-S and Pv-30R larvae when exposed to 300 ng/µL of dsSrp54k. We showed that the mortality of Pv-S larvae was achieved 100% after exposed to dsSrp54k for 7 days, while the mortality of Pv-30R under the same treatment was about 12%, indicating high-level resistance to dsSrp54k was established in Pv-30R (Fig. 2A). In consistent with this, the remarkable difference was also observed between the damaged area of the dsSrp54k-coated willow leaves caused by Pv-S and Pv-30R larvae after feeding with for 4 days. Although rarely little scale damage was observed in the dsSrp54k-coated willow leaves caused by Pv-S larvae, the dsSrp54k-coated willow leaves were almost completely consumed up by Pv-30R larvae (Fig. 2B), indicating the high viability of Pv-30R when exposed to dsSrp54k. The mean weight of Pv-S was significantly reduced after fed with dsSrp54k-coated willow leaves for 5 days compared with that of Pv-30R. No significant differences were detected in the Pv-30R larvae fed with and without dsSrp54k (Fig. 2C). Moreover, as expected, Srp54k expression was significantly (P = 0.0035) suppressed in Pv-S larvae fed with dsSrp54k-coated willow leaves compared with control group. In contrast, no such reduced expression (P = 0.282) of Srp54k was investigated in Pv-30R larvae fed with dsSrp54k-coated willow leaves compared with control group, indicating that feeding dsSrp54k was unable to induce the gene silencing in Pv-30R (Fig. 2D).

In order to determine the detailed resistance ratio of Pv-30R relative to Pv-S, bioassays for Pv-S and Pv-30R were conducted with a series concentrations of dsSrp54k. The median lethal concentration (LC₅₀) values for Pv-S was calculated as 0.073 (0.053, 0.093) ng/µL, while the LC₅₀ for Pv-30R was evaluated as more than 300 ng/µL. Since the mortality of Pv-30R under the highest tested concentration of dsSrp54k was less than 50% (Table 2), the resistance ratio of Pv-30R relative to Pv-S against dsSrp54k was determined as > 4110-fold.

Table 2

Relative resistance level of Pv-30R to the susceptible strain Pv-S.
Strain	dsRNA	LC₅₀^a (95%FL)^b	Slope ± SE	χ²	RR^c
Pv-S	dsSrp54k	0.073 (0.053–0.093)	2.792 ± 0.465	4.917	-
Pv-30R	dsSrp54k	> 300	-	-	> 4110
^a LC₅₀: median lethal concentration, ng/µL.
^b FL: fiducial interval.
^c RR: resistance ratio was calculated as LC₅₀ value of Pv-30R / LC₅₀ value of Pv-S.

3.2 Cross-resistance to other dsRNAs and Bt-Cry3Bb protein in Pv-30R

To determine whether Pv-30R exhibit cross-resistance to dsRNAs targeting other genes in P. versicolora, we tested Pv-30R with two dsRNAs targeting β-Actin and Snap in P. versicolora (Zhang et al. 2019). As expected from its high susceptibility, Pv-S larvae fed with dsActin and dsSnap were 100% killed after 6 and 8 days, respectively (Fig. 3A). In contrast, the mortality of Pv-30R were both less than 20% after feeding with dsActin and dsSnap for 8 days (Fig. 3A). The Pv-30R larvae were more active at feeding on the willow leaves coated with either dsActin or dsSnap, respectively. While the Pv-S larvae displayed food refusal behavior and caused little damage to the willow leaves coated with dsActin and dsSnap (Fig. 3B). Moreover, feeding dsActin and dsSnap resulted in significant reduction (P < 0.05, Tukey’s HSD multiple comparison test) of targeted gene expression compared with control groups in Pv-S larvae but not in Pv-30R larvae (Fig. 3C), indicating that dsActin and dsSnap induced the RNAi response and accounted for cross-resistance test in Pv-30R. These results support that Pv-30R exhibit strong cross-resistance to other dsRNAs than dsSrp54k.

To further test whether dsRNA-resistant P. versicolora larvae are resistant to insecticidal Bt protein, Pv-S and Pv-30R larvae were fed with the leaves from a Cry3Bb-transplastomic poplar (Pa-YY26) line (Xu et al. 2020). We showed that both Pv-S and Pv-30R were killed after 2 days of feeding (Fig. 4A and B). These data indicate Pv-30R is susceptible to insecticidal Cry3Bb protein as a similar level to that of Pv-S.

3.3 Genetics of the resistance to dsRNA in Pv-30R

To determine the inheritance characteristics of resistance to dsRNA in Pv-30R, reciprocal crosses between Pv-S and Pv-30R were conducted and the response of F1 generation larvae to dsSrp54k (30 ng/µL) was investigated. Although all Pv-30R larvae were able to survive at the tested concentration of dsSrp54k, the larvae from Pv-S and the F1 progeny from reciprocal crosses exhibited susceptible to the exposure of dsSrp54k and the survival rates of Pv-S and F1 progeny were 0 and 5%, respectively, after 7 days of feeding with dsSrp54k-coated willow leaves (Fig. 5). These results suggest that the resistance to dsRNA in Pv-30R is autosomal inheritance. The dominance value h was further calculated as 0.025, indicating the resistance to dsRNA in Pv-30R is incomplete recessive inheritance.

3.4 Potential mechanism of resistance to dsRNA in Pv-30R

To test whether resistance to dsRNA in Pv-30R could be associated with the degradation of dsRNA in larvae midguts, the midgut juice were collected from the third-instar larvae of Pv-S and Pv-30R, and incubated with dsRNA in vitro at 28°C. Results from integrity analysis of dsRNA showed that, no significant differences were observed between midgut juice from Pv-S and Pv-30R at any time point during the process of dsRNA degradation assays (Fig. 6), indicating that resistance to dsRNA in Pv-30R is unlikely associated with the dsRNA degradation capacity.

We finally examined whether resistance to dsRNA could be associated with the inability of the midgut cells to take up dsRNAs. To this end, we synthesized fluorescein-labeled dsGFP and incubated with midguts dissected from Pv-S and Pv-30R larvae. After 16 h of incubation, we discovered numerous dsGFP were localized inside Pv-S midgut cells, whereas almost no fluorescent signals were detected in Pv-30R midgut cells (Fig. 7). These results suggest resistance in Pv-30R could be associated with the impaired dsRNA uptake by midgut cells.

In this study, we report the first case of dsRNA-resistance in a forest pest P. versicolora by foliar delivery of insecticidal dsRNA. Similar to the previous study for resistance to dsRNA in Leptinotarsa decemlineata (Mishra et al. 2021), high-level resistance to RNAi was developed quickly under the chronic exposure to dsRNA in P. versicolora. Consistent with the selection procedure in L. decemlineata, the rapid evolution of resistance to dsRNA in P. versicolora may attributed to the increasing concentration of dsRNA during the whole selection process (Mishra et al. 2021). Also, use of the foliar delivery method can maintain P. versicolora under the insecticidal dsRNA stress throughout their whole life cycles. However, different with the initial generation of L. decemlineata for resistance selection that were collected from diverse locations in the USA (Mishra et al. 2021), the original source of high-level dsRNA-resistant P. versicolora was same as the susceptible strain, which was collected from one location in China and has been reared in laboratory for multiple generations without exposure to any insecticides. In addition to that, the number of P. versicolora for initial selection was less than that in the case of L. decemlineata. Therefore, these results support that individuals harboring resistance alleles in P. versicolora may be not rare in field population even at a relative small scale location (Chen et al. 2023).

Another potential factor may explains the rapid development of resistance to dsRNA in P. versicolora is the lack of fitness cost in Pv-30R. A recent study has confirmed that no fitness cost was detected in the dsRNA-resistant L. decemlineata (Pinto et al. 2023). Lack of fitness cost may result in the resistance alleles exist frequently and were stably maintained in field population (ffrench-Constant and Bass 2017; Kliot and Ghanim 2012). After the introduction of dsRNA, evolution of resistance would be expected relative quickly, which has been confirmed in selection of strains collected from the field population of D. v. virgifera and L. decemlineata. However, the fitness cost of Pv-30R in P. versicolora remains to be determined, even though no fitness cost has been reported in dsRNA-resistant insect until now.

Consistent with previously reported two cases in D. v. virgifera and L. decemlineata, resistance to dsRNA in Pv-30R was confirmed to be sequence-nonspecific, which could be considered as the hurdle for application of pyramiding of insecticidal dsRNAs against multiple genes (Zhu and Palli 2020). The broad spectrum resistance to multiple dsRNAs could be resulted from target-site mutation(s) in RNAi pathways for dsRNA uptake, processing of dsRNA into siRNA and spread of the RNAi signaling (Baum and Roberts 2014). To a certain extent, resistance to multiple dsRNAs in this study and the reported resistance to dsRNAs in D. v. virgifera and L. decemlineata should be substantially defined as resistance to RNAi, which is independent of the sequence of target gene and the characteristics of dsRNAs (Shukla et al. 2016). Due to the same modes of action mediating RNAi response for various dsRNAs in one pest species, it seems not helpful to delay or address the evolution of resistance to RNAi via alternating with additional various dsRNAs. In contrast, no cross-resistance between dsRNAs and Cry3B proteins were observed in Pv-30R, suggesting that pyramids of dsRNA and Cry protein could be available for the management of resistance in the field (Shaffer 2020). These results also supported that the modes of action underlying toxicity of insecticidal dsRNAs and Bt toxins are completely different, which has been confirmed in various species (Heckel 2020).

The refuge strategy has been the primary approach used worldwide to delay pest resistance to Bt crops and has been confirmed to be practically efficient in Bt resistance management (Hutchison et al. 2010; Tabashnik 2008). Three key factors favoring success of the refuge strategy are recessive inheritance of resistance, low resistance allele frequency and abundant refuges (Tabashnik and Carriere 2017). In current study, the inheritance of resistance to dsRNAs in Pv-30R was shown to be recessive, suggesting that the refuge strategy should be promising to manage resistance to dsRNAs in the field. Even though the possibility that resistance to dsRNA is non-recessive cannot be excluded, increasing refuge abundance can still substantially delay resistance based on the previous modeling results from Bt resistance (Tabashnik et al. 2013).

Degradation by dsRNases in midgut lumen and inefficient uptake by midgut epithelial cells have been known as the primary barriers to efficiency of RNAi-mediated pest control (Choudhary et al. 2021; Cooper et al. 2019). Cross-resistance to other dsRNAs observed in Pv-30R suggested a potential mechanism of resistance involved in processes of dsRNA degradation and uptake of dsRNA in larvae midgut (Cooper et al. 2019). Although we found that no remarkable differences in the degradation efficiency between the Pv-S and Pv-30R strain, the uptake of dsRNA in Pv-30R was shown to be decreased compared with that in Pv-S, which was in line with the results documented in D. v. virgifera (Khajuria et al. 2018). These data also supported the cross-resistance to additional dsRNAs other than dsSrp54k in Pv-30R. Together evidences from the case of D. v. virgifera and P. versicolora in current study, defect in uptake of dsRNA could be a common mechanism of resistance to dsRNA or environmental RNAi, even though the potential mechanism of resistance to dsRNA in the CEAS 300 and Pv-30R strains was still unclear (Mishra et al. 2021).

Overall, this study demonstrates high-level resistance to dsRNA can rapidly evolve in the forest pest insect. In general, cross-resistance to dsRNAs would be a hurdle for the application of resistance management tool that based on alternate dsRNA targeting the same species. The refuge strategy and pyramids of Bt protein and dsRNA should be preferentially taken into account to delay the potential rapid evolution of dsRNA resistance in the field. Taken together with the other two cases of resistance to dsRNA, a common mechanism is supposed to be shared to drive the evolution of resistance to dsRNA in various coleopteran insects.

Acknowledgments

This research was funded by grants from the National Natural Science Foundation of China (32271912)

Author Contributions: J.Z. designed research; C.L. and M.Z. performed research; C.L., M.Z. and J.Z. analyzed data; C.L. and J.Z. wrote the paper with input from other authors.

Funding

This research was funded by grants from the National Natural Science Foundation of China (32271912).

Competing interests

The authors declare no conflict of interest.

Human and animal participants

This article does not contain any studies with human participants or animals performed by any of the authors.

Agrawal N, Dasaradhi PV, Mohmmed A et al. (2003) RNA interference: biology, mechanism, and applications Microbiol Mol Biol Rev 67:657-685 doi:10.1128/MMBR.67.4.657-685.2003
Baum JA, Bogaert T, Clinton W et al. (2007) Control of coleopteran insect pests through RNA interference Nat Biotechnol 25:1322-1326 doi:10.1038/nbt1359
Baum JA, Roberts JK (2014) Progress towards RNAi-mediated insect pest management Adv Insect Physiol 47:249-295 doi:10.1016/B978-0-12-800197-4.00005-1
Chen YH, Cohen ZP, Bueno EM et al. (2023) Rapid evolution of insecticide resistance in the Colorado potato beetle, Leptinotarsa decemlineata Curr Opin Insect Sci 55:101000 doi:10.1016/j.cois.2022.101000
Choudhary C, Meghwanshi KK, Shukla N et al. (2021) Innate and adaptive resistance to RNAi: a major challenge and hurdle to the development of double stranded RNA-based pesticides 3 Biotech 11:498 doi:10.1007/s13205-021-03049-3
Cooper AM, Silver K, Zhang J et al. (2019) Molecular mechanisms influencing efficiency of RNA interference in insects Pest Manag Sci 75:18-28 doi:10.1002/ps.5126
Cullen BR (2014) Viruses and RNA interference: issues and controversies J Virol 88:12934-12936 doi:10.1128/JVI.01179-14
ffrench-Constant RH, Bass C (2017) Does resistance really carry a fitness cost? Curr Opin Insect Sci 21:39-46 doi:10.1016/j.cois.2017.04.011
Fire A, Xu SQ, Montgomery MK et al. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans Nature 391:806-811 doi:Doi 10.1038/35888
Fletcher SJ, Reeves PT, Hoang BT et al. (2020) A perspective on RNAi-based biopesticides Front Plant Sci 11:51 doi:10.3389/fpls.2020.00051
Gassmann AJ, Onstad DW, Pittendrigh BR (2009) Evolutionary analysis of herbivorous insects in natural and agricultural environments Pest Manag Sci 65:1174-1181 doi:10.1002/ps.1844
Heckel DG (2020) How do toxins from Bacillus thuringiensis kill insects? An evolutionary perspective Arch Insect Biochem 104:e21673 doi:10.1002/arch.21673
Hutchison WD, Burkness EC, Mitchell PD et al. (2010) Areawide Suppression of European Corn Borer with Bt Maize Reaps Savings to Non-Bt Maize Growers Science 330:222-225 doi:10.1126/science.1190242
Jurat-Fuentes JL, Heckel DG, Ferre J (2021) Mechanisms of resistance to insecticidal proteins from Bacillus thuringiensis Annu Rev Entomol 66:121-140 doi:10.1146/annurev-ento-052620-073348
Khajuria C, Ivashuta S, Wiggins E et al. (2018) Development and characterization of the first dsRNA-resistant insect population from western corn rootworm, Diabrotica virgifera virgifera LeConte PLoS One 13:e0197059 doi:10.1371/journal.pone.0197059
Kliot A, Ghanim M (2012) Fitness costs associated with insecticide resistance Pest Manag Sci 68:1431-1437 doi:10.1002/ps.3395
Liu Y, Tabashnik BE (1997) Inheritance of resistance to the Bacillus thuringiensis toxin Cry1C in the Diamondback moth Appl Environ Microbiol 63:2218-2223 doi:10.1128/aem.63.6.2218-2223.1997
Mishra S, Dee J, Moar W et al. (2021) Selection for high levels of resistance to double-stranded RNA (dsRNA) in Colorado potato beetle (Leptinotarsa decemlineata Say) using non-transgenic foliar delivery Sci Rep 11:6523 doi:10.1038/s41598-021-85876-1
Nandety RS, Kuo YW, Nouri S et al. (2015) Emerging strategies for RNA interference (RNAi) applications in insects Bioengineered 6:8-19 doi:10.4161/21655979.2014.979701
Ni M, Ma W, Wang X et al. (2017) Next-generation transgenic cotton: pyramiding RNAi and Bt counters insect resistance Plant Biotechnol J 15:1204-1213 doi:10.1111/pbi.12709
Niu L, Yan H, Sun Y et al. (2022) Nanoparticle facilitated stacked-dsRNA improves suppression of the Lepidoperan pest Chilo suppresallis Pestic Biochem Physiol 187:105183 doi:10.1016/j.pestbp.2022.105183
Pinto MMD, Ferreira Dos Santos R, De Bortoli SA et al. (2023) Lack of fitness costs in dsRNA-resistant Leptinotarsa decemlineata ([Coleoptera]: [Chrysomelidae]) J Econ Entomol:1–8 doi:10.1093/jee/toad095
Price DRG, Gatehouse JA (2008) RNAi-mediated crop protection against insects Trends Biotechnol 26:393-400 doi:10.1016/j.tibtech.2008.04.004
Rodrigues TB, Mishra SK, Sridharan K et al. (2021) First sprayable double-stranded RNA-based biopesticide product targets proteasome subunit beta Type-5 in Colorado potato beetle (Leptinotarsa decemlineata) Front Plant Sci 12:728652 doi:10.3389/fpls.2021.728652
Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C-T method Nat Protoc 3:1101-1108 doi:DOI 10.1038/nprot.2008.73
Shaffer L (2020) Inner Workings: RNA-based pesticides aim to get around resistance problems Proc Natl Acad Sci U S A 117:32823-32826 doi:10.1073/pnas.2024033117
Shukla JN, Kalsi M, Sethi A et al. (2016) Reduced stability and intracellular transport of dsRNA contribute to poor RNAi response in lepidopteran insects RNA Biol 13:656-669 doi:10.1080/15476286.2016.1191728
Suzuki T, Nunes MA, Espana MU et al. (2017) RNAi-based reverse genetics in the chelicerate model Tetranychus urticae: A comparative analysis of five methods for gene silencing PLoS One 12:e0180654 doi:10.1371/journal.pone.0180654
Tabashnik BE (2008) Delaying insect resistance to transgenic crops Proc Natl Acad Sci U S A 105:19029-19030 doi:10.1073/pnas.0810763106
Tabashnik BE, Brevault T, Carriere Y (2013) Insect resistance to Bt crops: lessons from the first billion acres Nat Biotechnol 31:510-521 doi:10.1038/nbt.2597
Tabashnik BE, Carriere Y (2017) Surge in insect resistance to transgenic crops and prospects for sustainability Nat Biotechnol 35:926-935 doi:10.1038/nbt.3974
Xu L, Xu S, Sun L et al. (2021) Synergistic action of the gut microbiota in environmental RNA interference in a leaf beetle Microbiome 9:98 doi:10.1186/s40168-021-01066-1
Xu S, Zhang Y, Li S et al. (2020) Plastid-expressed Bacillus thuringiensis (Bt) cry3Bb confers high mortality to a leaf eating beetle in poplar Plant Cell Rep 39:317-323 doi:10.1007/s00299-019-02492-0
Zhang J, Khan SA, Hasse C et al. (2015) Pest control. Full crop protection from an insect pest by expression of long double-stranded RNAs in plastids Science 347:991-994 doi:10.1126/science.1261680
Zhang YQ, Xu LT, Li SC et al. (2019) Bacteria-mediated RNA interference for management of Plagiodera versicolora (Coleoptera: Chrysomelidae) Insects 10:415 doi:ARTN 415 10.3390/insects10120415
Zhu KY, Palli SR (2020) Mechanisms, applications, and challenges of insect RNA interference Annu Rev Entomol 65:293-311 doi:10.1146/annurev-ento-011019-025224

No competing interests reported.

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published 24 Apr, 2024

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Characterization and potential mechanism of resistance to double-stranded RNA in willow leaf beetle, Plagiodera versicolora

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Abstract

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Key Message

1 Introduction

2 Materials and methods

2.1 Insect rearing

2.2 In vitro dsRNA synthesis

2.3 Selection for resistance to RNA pesticides

2.4 Evaluating for crossresistance to other dsRNAs and Bt Cry3Bb protein

2.5 Evaluating inheritance of resistance to dsRNA

2.6 RNA extraction, cDNA synthesis and qRT-PCR analysis

2.7 Investigation of degradation and uptake of dsRNA in the larvae midgut.

2.8 Statistical analysis

3 Results

3.1 Selection for resistance to dsRNA in P. versicolora

3.2 Cross-resistance to other dsRNAs and Bt-Cry3Bb protein in Pv-30R

3.3 Genetics of the resistance to dsRNA in Pv-30R

3.4 Potential mechanism of resistance to dsRNA in Pv-30R

4 Discussion

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References

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Supplementary Files

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