You are currently viewing a new version of our website. To view the old version click .
MDPIMDPI
Search all articles
Insects
Download PDF
  • Article
  • Open Access

16 October 2025

RNAi of vATPasea Affects Survival and Larval-Pupal Development in Plutella xylostella

,
,
,
,
,
,
,
,
and
1
Key Laboratory of Agricultural Genetically Modified Organisms Traceability, Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, Ministry of Agriculture and Rural Affairs, Wuhan 430062, China
2
Enshi Tujia and Miao Autonomous Prefecture Academy of Agricultural Sciences, Enshi 445000, China
3
Department of Chemical Technology of Silicate Materials and Rare, Tashkent Institute of Chemical Texnology, Tashkent 100011, Uzbekistan
*
Authors to whom correspondence should be addressed.
Insects2025, 16(10), 1054;https://doi.org/10.3390/insects16101054
This article belongs to the Special Issue Advances in Insect Pest Management: Innovative Approaches to Enhance Plant Protection
Version Notes
Order Reprints

Simple Summary

Relatively little attention has been given to genes encoding V0 subcomplex subunits. In the study, we identified PxvATPasea specifically and evaluated expression profiles of PxvATPasea across developmental stages and among tissues. By injecting two doses of dsPxvATPasea (800 ng or 1200 ng) in Plutella xylostella, we showed that PxvATPasea is a potential molecular target gene and RNAi efficiencies worked in a dose-dependent way.

Abstract

The diamondback moth, Plutella xylostella, is a major pest of brassica vegetables and oilseed crops, posing a serious threat to China’s grain and oil production. RNA interference (RNAi) has been developed as an efficient strategy to control pests. In this study, the effects of RNAi on P. xylostella were evaluated by injecting two doses of synthesized dsPxvATPasea. The transcripts of PxvATPasea were widely transcribed during different developmental stages from egg to adult. They were abundantly expressed in the hindgut and Malpighian tubules, compared with other tissue types. Introduction of 800 ng dsPxvATPasea in the fourth-instar larvae greatly reduced corresponding mRNA levels by 3.1 and 1.4 times on day 2 and 3, respectively, causing 66.6% mortality and 33.4% treated larvae pupated. Silencing PxvATPasea by injecting 1200 ng dsRNA significantly decreased the expression level by 5.0 and 2.0 times on the second and third day, leading to 79.2% larval lethality and 20.8% depleted larvae pupated. Moreover, introducing 800 ng or 1200 ng dsPxvATPasea finally reduced larval fresh weight by 22.1% and 28.8%, respectively. The results indicated that the silencing efficiency of PxvATPasea worked in a dose-dependent way. Consequently, PxvATPasea is a potential molecular target gene. Our findings will facilitate the application of RNAi technology to manage P. xylostella.

1. Introduction

The diamondback moth, Plutella xylostella, is one of the primary lepidopteran pests attacking brassica vegetables and oilseed crops, causing monetary losses of around US $4–5 billion worldwide [1,2]. At present, chemical pesticides are applied mainly to manage this pest, such as chlorantraniliprole, chlorpyrifos, and indoxacarb [3]. However, owing to the rapid development of resistance, it is more and more difficult to control its field populations based on insecticides [2]. Thus, it seems much more urgent and necessary to develop an efficient strategy to manage the population of P. xylostella.
RNA interference (RNAi) is an efficient molecular mechanism for suppressing the expression of post-transcriptional target genes triggered by double-stranded RNA (dsRNA), which can be used to develop an environmentally friendly and reduced-risk insect pest management approach [4,5,6,7]. In recent years, numerous research have demonstrated that RNAi-based integrated pest management works well in various insects [7,8,9,10,11,12,13]. For example, use plant-mediated RNA interference strategy to control Frankliniella occidentalis [9] and Apolygus lucorum [10]; RNAi silencing protease genes can affect mortality in Helicoverpa armigera [12]; continuous ingestion of dsLdRan can kill the larvae and adults of Leptinotarsa decemlineata [7]; knockdown of HvUAP impairs larval growth in Henosepilachna vigintioctopunctata [13]. Currently, silencing target genes PxPiwi [14] and PxTH [15] in P. xylostella affected larval development. Thus, it seems practicable to apply RNAi-based methods to manage population of P. xylostella.
Vacuolar-type H+-ATPase (vATPase) is a proton translocating pump localized in the internal membranes of eukaryotes, functioning by hydrolyzing ATP to ADP and phosphate to pump protons across membranes [16,17,18,19]. The structures of vATPases are evolutionarily conserved in eukaryotes, consisting of two subcomplexes, V1 and V0 [20,21]. The V1 subcomplex contains eight different subunits (A through H), with stoichiometry of A3B3CDE3FG3H in Manduca sexta and Saccharomyces cerevisiae, which is related to ATP hydrolysis. The V0 subcomplex consists of five different subunits (a1d1c4–5c′1c″1) and accessory proteins, which can drive transmembrane proton movement [21,22,23,24,25,26].
Currently, relatively little attention has been given to genes encoding V0 subcomplex subunits, and only two research have verified that depletion of vATPasea was lethal [27,28]. In Drosophila melanogaster, P-element insertions in vha100-2 are lethal [27]. In H. vigintioctopunctata, knockdown of HvvATPasea in the third-instar larvae caused 100% larval lethality [28]. It is vital to determine whether silencing genes encoding V0 subcomplex subunits results in severe defective phenotypes in other insects. In addition, compared to Bt-transgenic Brassica napus, expression of CHS1 hpRNA in transgenic B. napus shortened the mortality time in P. xylostella [1]. Therefore, RNAi-based control of Plutella xylostella populations by feeding them dsRNA targeting PxvATPasea holds promise for future development.
In the present study, PxvATPasea was chosen as the target gene to assess the possibility of management methods for P. xylostella. Specifically, we (i) cloned, aligned, and phylogenetically analyzed the vATPasea gene in P. xylostella; (ii) evaluated expression profiles of PxvATPasea across developmental stages and among tissues; (iii) assessed the RNAi efficiency by measuring the suppression of PxvATPasea expression after treatment with synthesized dsPxvATPasea in vitro; and (iv) examined the effects on larval weight, survival rate, pupation rate, and emergence rate following injection of dsPxvATPasea.

2. Materials and Methods

2.1. Insect Rearing

P. xylostella was collected from B. napus in Haidong City, Qinghai Province, China. The larvae of P. xylostella were fed on fresh brassica leaves at 26 ± 1 °C, 16 h:8 h photoperiod, and 60% ± 5% relative humidity. The adults were reared using 10% honey solution as food.

2.2. Molecular Cloning

The putative PxvATPasea was obtained from the genome and transcriptome data of P. xylostella [29,30]. The correctness of the sequence was proven by polymerase chain reaction (PCR) using primers in Table S1 and Figure S1. The accession number of sequenced cDNA was XP_048479200.1.
Phylogenetic analysis of PxvATPasea and other vATPasea sequences downloaded from NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 18 April 2024) was conducted using MEGA 5.0 software (https://sourceforge.net/projects/mega5/, accessed on 18 April 2024) and the neighbor-joining method, with 1000 bootstrap replications.

2.3. Synthesis of dsRNA Molecules

The two cDNA fragments targeting PxvATPasea and enhanced green fluorescent protein were amplified by PCR using special primers containing the T7 promoter sequence (Table S1, Figure S1). To ensure the specificity and efficacy of the dsRNA, the target sequences by BioEdit 7.0 software were subjected to BLASTN analysis against the P. xylostella transcriptome and evaluated using a web-based prediction tool (https://www.dsrna-engineer.cn, accessed on 18 April 2024) for on-target and off-target sites. Regions with a high density of on-target sites were selected while avoiding potential off-target sequences, thereby excluding any segments with ≥20 bp identical matches that could lead to off-target effects (Figure S2). dsRNA was then synthesized using the MEGAscript T7 High Yield Transcription Kit (Ambion, Austin, TX, USA), according to the manufacturer’s instructions. The reaction mixture contained 2 μL of T7 Express Enzyme Mix (Promega, Beijing, China), 10 μL of RiboMAX™ Express T7 2× Buffer (Promega, Beijing, China), and 1 μg of DNA template and was incubated at 37 °C for 2–6 h, followed by 70 °C for 10 min, and slowly cooled to room temperature (approximately 20 min) for dsRNA annealing. The DNA template and single-stranded RNA were removed by treatment with DNase and RNase A, respectively, and the dsRNA product was purified using a gel extraction kit (Omega Bio-tek, Norcross, GA, USA). The yield of dsRNA was determined by measuring absorbance at 260 nm with a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and its integrity was verified by agarose gel electrophoresis. The purified dsRNA was aliquoted and stored at −80 °C until use.

2.4. Introduction of dsRNA

The same method as previously described was used to inject dsRNA [31,32,33]. Briefly, newly molted fourth-instar larvae were selected for microinjection. Borosilicate microcapillaries were pulled using a PC-10 puller (Narishige, Tokyo, Japan). A volume of 200 nL containing one of two dsRNA doses (800 ng or 1200 ng in total, based on references [34]) was delivered into the larval hemolymph using a microinjection system from World Precision Instruments (Sarasota, FL, USA). Larvae injected with dsGFP served as the negative control. A group of eight injected larvae constituted one replicate, and each dsRNA treatment was repeated six times. After injection, larvae were transferred to individual plastic rearing boxes and fed with fresh rape leaves. For sampling, three replicates were collected 2 and 3 days post-injection for qRT-PCR analysis to assess RNAi efficiency. Another three replicates were maintained for a 3-week observation period to monitor defective phenotypes, changes in larval weight, survival rate, pupation rate, and emergence rate.

2.5. Real-Time Quantitative PCR (qRT-PCR)

RNA templates of samples were collected to analyze the temporal expression profiles and tissue transcription patterns. Moreover, for testing the effects of the treatments, total RNA was extracted from treated larvae by the TRIzol reagent (YiFeiXue Tech, Nanjing, China). Each sample contained eight individuals and was repeated three times. Quantitative real-time PCRs (qRT-PCR) were conducted to assess the transcript expression levels according to the previously described method [35]. The qRT-PCR was performed with three biological and technical replicates. The relative mRNA expression levels were analyzed using 2−ΔΔCt method.

2.6. Data Analysis

We used The SPSS Statistics 27 software for Windows (Chicago, IL, USA) for the statistical analyses. The averages (±SE) were used to analyze variance with the Tukey–Kramer test. Survival curves were analyzed using a log-rank test (Mantel–Cox, 95% CI) in GraphPad Prism software version 8.0.

3. Results

3.1. Identification of PxvATPasea

A putative full-length cDNA encoding PxvATPasea was obtained in P. xylostella by mining transcriptome data. The cDNA was comprised of a 2520 bp complete open reading frame encoding 839 amino acid residues (Figure S1).
The sequences of vATPasea proteins in different insects were highly conserved. It contained V_ATPase_I structural domain (Figure 1A). The phylogenetic dendrogram of vATPasea proteins from 12 species was built to assess the evolutionary relationships (Figure 1B). These sequences were from four Lepidoptera, Pieris rapae, H. armigera, Bombyx mori, and P. xylostella; two Hymenoptera, Apis mellifera and Solenopsis Invicta; two Diptera, Aedes aegypti and Drosophila willistoni; two Hemiptera, Nilaparvata lugens and Myzus persicae; and two Coleoptera, Leptinotarsa decemlineata and Anoplophora glabripennis. The unrooted tree revealed that the vATPasea-like proteins of species from the same order were clustered together. Apparently, vATPasea from P. xylostella belonged to Lepidoptera subclade (Figure 1B).
Figure 1. Alignment (A) and phylogenetic analysis (B) of vATPase subunits a (PxvATPasea) derived from Plutella xylostella. (A) The proteins originate from Pieris rapae (Pr), Nilaparvata lugens (Nl), Helicoverpa armigera (Ha), Drosophila willistoni (Dw), Apis mellifera (Am), and Plutella xylostella (Px). Increasing background intensity (from light to dark) indicates an increase in sequence similarity. Gaps are introduced to permit alignment. (B) vATPasea proteins are from four Lepidoptera, Bombyx mori, Pieris rapae, Plutella xylostella, and Helicoverpa armigera; two Hymenoptera, Apis mellifera and Solenopsis Invicta; two Diptera, Aedes aegypti and Drosophila willistoni; two Hemiptera, Nilaparvata lugens and Myzus persicae; and two Coleoptera, Leptinotarsa decemlineata and Anoplophora glabripennis. The tree is constructed using the neighbor-joining method based on the full-length protein sequence alignments. Bootstrap analyses of 1000 replications are carried out, and bootstrap values > 60% are shown on the tree.

3.2. The Expression Profiles of PxvATPasea

We used qRT-PCR to test the temporal expression pattern of PxvATPasea. The mRNA levels of PxvATPasea were widely transcribed during different developmental stages from embryo (egg) to adult. Its mRNA levels rose to a peak at day 2 of third-instar larval period; however, the lowest PxvATPasea level was detectable at the late fourth-instar larvae (day 2 and day 3). It was clearly observed that the mRNA levels of PxvATPasea regularly aggrandized during ecdysis period (Figure 2A).
Figure 2. Temporal (A) and tissue (B) expression profiles of PxvATPasea in Plutella xylostella. For temporal transcription profile test, complementary DNA templates were derived from the eggs, first through fourth-larval instars at an interval of 1 day (1st indicated 0 and 1 days of first-instar larvae, D0 indicated newly ecdysed larvae or pupae, or D0 indicated newly ecdysed larvae), pupae (newly ecdysed pupae), and adults (newly emerged adults). For tissue expression pattern test, templates were from the head, foregut (FG), midgut (MG), hindgut (HG), hemolymph (HE), and epidermis (EP) of the day 4 fourth-instar larvae. For each sample, three independent pools of 5–10 individuals were measured in technical triplicate using qRT-PCR. The values were calculated using the 2−ΔΔCT method. The lowest transcript levels at egg and head are set as 1. The columns represent averages with vertical lines indicating SE.
The spatial expression profiles of PxvATPasea were evaluated in all tested tissues, including head, foregut, midgut, hindgut, hemolymph, Malpighian tubules, and epidermis. The mRNA levels of PxvATPasea were high in the hindgut and Malpighian tubules, intermediate in the foregut and midgut, and low in the hemolymph, epidermis, and head (Figure 2B).

3.3. Effects of RNAi for 800 ng of dsPxvATPasea at the Fourth-Instar Larvae

To evaluate the effects of knockdown of PxvATPasea in P. xylostella, we silenced PxvATPasea by injection of dsRNA. Introduction of 800 ng dsPxvATPasea in the newly molted fourth-instar larvae significantly decreased the PxvATPasea mRNA level by 3.1 and 1.4 times at 2 d and 3 d, respectively (Figure 3A).
Figure 3. Effects of PxvATPasea silencing in fourth-instar larvae of Plutella xylostella. Newly molted fourth-instar larvae were injected with 0.2 µL of solution containing 800 ng of dsPxvATPasea, with an equivalent amount of dsGFP serving as the negative control. Injected larvae were subsequently transferred to fresh rape leaves for rearing. Expression levels of PxvATPasea were measured at 48 and 72 h post-injection (A). Relative transcript levels are presented as the ratio of relative copy numbers in treated larvae to those in the dsGFP control group (set as 1). Larval weight (B), survival rate (C), pupation rate (D), and emergence rate (E) were recorded over a 5-day trial period. Data are presented as mean ± SE. Different letters indicate statistically significant differences at p < 0.05.
Knockdown of PxvATPasea significantly restrained the development of the larvae, with fresh weights reduced by 26.0% and 22.1% at day 3 and 4, respectively, compared to the dsGFP control (Figure 3B). RNAi of PxvATPasea caused a significant mortality of the larvae. After the bioassay, mortality was 16.7%, 37.5%, and 66.6% at day 2, 3, and 4, respectively, in this treatment (Figure 3C). The remaining 33.4% larvae successfully pupated (Figure 3D). There were no significant differences in emergence rate among the two groups (Figure 3E). The dsPxvATPasea-treated larvae possessed small and misshapen body size. Subsequently, they slowly withered, blackened, and, lastly, died (Figure 4A vs. Figure 4B).
Figure 4. Impacts on knockdown of PxvATPasea in the fourth-instar larvae of Plutella xylostella. Phenotypic impacts of vATPasea silencing via dsRNA injection were assessed through developmental observation. Larvae were injected with solutions containing either 800 ng or 1200 ng of dsPxvATPasea, with an equivalent amount of dsGFP serving as the negative control. After injection, the larvae were transferred to rape leaves for rearing and monitored at 24 h intervals for developmental delays. Phenotypic documentation of both the dsPxvATPasea-treated and dsGFP control groups was performed on the third day after the start of the experiment.

3.4. Impacts on Introduction of 1200 ng dsPxvATPasea at the Fourth-Instar Larvae

Introduction of 1200 ng dsPxvATPasea by microinjection at the newly molted fourth-instar larvae successfully reduced the mRNA level of PxvATPasea by 5.0 and 2.0 times at 2 d and 3 d, respectively, compared to the dsGFP-treated group (Figure 5A).
Figure 5. Effects of different doses of dsRNA on the suppression of PxvATPasea in Plutella xylostella. Newly molted fourth-instar larvae were injected with 0.2 µL of solution containing 1200 ng of dsPxvATPasea, with dsGFP injection used as the negative control. Under identical experimental conditions, PxvATPasea expression levels (A), weight (B), survival rate (C), pupation rate (D), and emergence rate (E) were analyzed. Data are shown as mean ± SE. Different letters denote significant differences at p < 0.05.
RNAi of PxvATPasea significantly inhibited the larval growth (Figure 5B), with fresh weights decreased by 28.0%, 30.0%, and 28.8% on day 2, 3, and 4, respectively, compared to the dsGFP-injected group (Figure 5B). In addition, silencing PxvATPasea seriously led to larval lethality, with mortality at 25.0%, 66.6%, and 79.2%, at day 2, 3, and 4, respectively (Figure 5C). The remaining 20.8% larvae successfully pupated (Figure 5D), and the emergence rate showed no significant differences (Figure 5E). The PxvATPasea RNAi larvae failed to molt their cuticle to become pupae. Their body size was small and abnormal, compared with the dsGFP-injected group. Subsequently, these stunting PxvATPasea hypomorphs became withered, dried, and blackened slowly and eventually died (Figure 4C vs. Figure 4D).

4. Discussion

Currently, relatively little attention has been given to genes encoding V0 subcomplex subunits. In the present study, we identified a vATPase subunit a gene in P. xylostella. We uncovered that silencing PxvATPasea affects the survival and growth of larvae.

4.1. RNAi Is a Powerful Tool to Manage P. xylostella

RNAi is one of the most important technologies in the last century [36], which is used as a powerful tool to study the functions of target genes or screen lethal genes for pest management in environmentally friendly and species-specific way [5,37,38,39,40]. Moreover, the development and application of biopesticides based on RNAi is a popular subject in pest management [41]. Up to date, great strides have been made in using genetically modified crops to control virous pests, including A. lucorum [10], L. decemlineata [42], Adelphocoris suturalis [43], Sitobion avenae [44], M. persicae [45], and so on. This evidence has strongly indicated that an RNAi-based strategy is viable to control pests.
The first step is to select suitable candidate genes for the development of RNAi-mediated control methods. Numerous studies have demonstrated that housekeeping genes such as actins [46]; cellular function genes like vacuolar ATPase [47]; energy metabolism genes, including eukaryotic translation initiation factor 5A [48]; and 20E signaling network genes like ecdysone receptors (EcR) [49,50,51] are known to be necessary for the growth of larvae. They can be developed as dsRNA insecticides or transgenic crops expressing dsRNA to control pests.
At present, RNAi of PxPiwi [14] and PxTH [15] in P. xylostella affected larval growth. Therefore, it seems very feasible to apply RNAi technology to control population of this pest.

4.2. PxvATPasea Is a Potential Candidate Gene to Control the Larvae of P. xylostella

In short, our results indicated that dsPxvATPasea is a potential candidate gene to control the larvae of P. xylostella. Firstly, vATPasea is a highly conserved evolutionarily ancient enzyme with the conserved domain of V_ATPase_I (Figure 1), which performs a wide range of functions in eukaryotic species [16,17,18,22,52].
Secondly, PxvATPasea was abundantly transcribed during different developmental stages, from embryo (egg) to adult. The expression of PxvATPasea was high at day 2 of the third-instar larval period and was low in the late fourth-instar larvae. Consistent with our results, the mRNA level of HvvATPasea was high in the third-instar larvae in H. vigintioctopunctata.
Thirdly, vATPases have an important role in nutrient absorption in the gut of various insects [19,53,54]. Our results showed that PxvATPasea was abundantly transcribed in the hindgut and Malpighian tubules in P. xylostella. Likewise, in H. vigintioctopunctata, the expression levels of HvvATPasea were highly expressed in hindgut [28].
Lastly, we uncovered that the introduction of dsPxvATPasea by injecting two different doses of dsRNA at the fourth-instar larvae significantly decreased the mRNA levels of PxvATPasea at different times, severely affected larval development, and killed 66.6% and 79.2% of larvae, respectively, under 800 ng and 1200 ng conditions (Figure 3 and Figure 4). In line with our result, in D. melanogaster, P-element insertions in vha100-2 are lethal [27]; RNAi of HvvATPasea in the third-instar larvae of H. vigintioctopunctata led to 100% larval lethality [28].
Consequently, PxvATPasea is a potential candidate gene to manage by RNAi-based management strategies in P. xylostella.
Current functional studies on insect vATPases have primarily focused on subunits of the V1 complex, while research on V0 complex subunits remains relatively limited. Silencing of vATPase subunit a in the migratory locust (Locusta migratoria) specifically affects the midgut epithelium and causes high mortality, mainly by reducing the number of columnar epithelial cells during midgut development, which subsequently lowers nutrient transport efficiency and leads to reduced body weight [55]. Similarly, RNAi-mediated knockdown of the vATPase-a2 gene in the Sogatella furcifera impacts feeding behavior, resulting in significant changes in mortality, phenotypic defect incidence, and adult eclosion rate [56]. In Lepidoptera, research on the vATPase a subunit remains largely unexplored. Current evidence demonstrates that knockdown of vATPasea in the diamondback moth reduces body weight and causes high mortality, consistent with findings from RNAi targeting vATPasea subunits in other insects. However, the precise mechanisms through which it influences vital physiological processes require further investigation.

4.3. Enhancing RNAi Efficiency by Stabilizing dsRNA in Insects

Multiple factors influence RNAi efficiency, with dsRNA stability being a critical determinant. Naked dsRNA introduced into insects via injection or feeding is susceptible to degradation by nucleases present in saliva, hemolymph, and the gut, which partially accounts for the low RNAi efficiency observed in certain insect species. Particularly in Lepidoptera, nucleases identified as RNAi efficiency-related nucleases (REases) have been recognized [57]. Studies demonstrate that insect hemolymph and gut fluid contain double-stranded ribonucleases (dsRNases), which are considered a major limiting factor for RNAi efficiency due to their impact on dsRNA stability in bodily fluids. In insects, reducing dsRNase activity helps minimize intracellular dsRNA degradation and enhances RNAi efficacy [58,59].
Regarding dsRNA degradation prevention, encapsulating dsRNA with protective materials effectively preserves its integrity. The use of nanoparticle complexes to encapsulate dsRNA represents a novel delivery strategy for RNAi-based applications. These diverse complexes facilitate improved cellular uptake of dsRNA due to their high transduction efficiency and low cytotoxicity, while also reducing the risk of nuclease degradation under environmental conditions, such as temperature variations and medium changes [60]. The most extensively tested nanocarriers include chitosan, liposomes, star polycations (SPcs), layered double hydroxides (LDH), and guanylated polymers (GNP). The control efficacy of different vATPase subunits in S. furcifera was evaluated using star polymer (SPc) nanoparticles via spray-induced and nanoparticle-delivered gene silencing (SI-NDGS), which successfully reduced target mRNA levels and vATPase enzyme activity, while also assessing the environmental safety of nanoparticle-encapsulated dsRNA [56]. Three distinct nanoparticle-encapsulated dsRNA complexes significantly knocked down multiple genes, including VATPase, in Earias vittella [61]. Loading dsRNA of the VATPaseA gene onto layered double hydroxide (LDH) enhanced its environmental stability, leading to mortality in Holotrichia parallela larvae and disruption of their cuticle and midgut structures [62]. Therefore, further research on RNAi approaches targeting dsPxvATPasea will represent a promising strategy for managing diamondback moth populations.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/insects16101054/s1, Table S1: A list of primers used for RT-PCR of the genes; Figure S1: A display of nucleic acid sequences of PxvATPasea from Plutella xylostella; Figure S2. On-target/off-target prediction and high-risk off-target regions of dsvATPasea.

Author Contributions

Experiments were designed by X.Y. (Xuetao Yu), J.L., L.L., G.W., X.Y. (Xiaohong Yan), and C.S. Figures designed and created by X.Y. (Xuetao Yu), J.L., L.L., G.W., X.Y. (Xiaohong Yan), and C.S. Statistical analyses were implemented by X.Y. (Xuetao Yu), J.L., L.L., G.W., L.Z., S.W., K.Y., X.W., Y.W., B.A., G.W., X.Y. (Xiaohong Yan), and C.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Key Research and Development Program of China (grant number 2024YFD1400800), the Outstanding Young Talent Cultivation Project of Oil Crops Research Institute of Chinese Academy of Agricultural Sciences (OYTCP-OCRI-2025-09), and the Innovation Project of Chinese Academy of Agricultural Sciences (No. 2060302-049-091).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Deng, P.; Peng, Y.; Sheng, Z.; Li, W.; Liu, Y. RNAi silencing CHS1 gene shortens the mortality time of Plutella xylostella feeding Bt-transgenic Brassica napus. Pest Manag. Sci. 2024, 80, 2610–2618. [Google Scholar] [CrossRef] [PubMed]
  2. Shehzad, M.; Bodlah, I.; Siddiqui, J.A.; Bodlah, M.A.; Fareen, A.G.E.; Islam, W. Recent insights into pesticide resistance mechanisms in Plutella xylostella and possible management strategies. Environ. Sci. Pollut. Res. Int. 2023, 30, 95296–95311. [Google Scholar] [CrossRef] [PubMed]
  3. Wang, M.; Zhu, B.; Zhang, L.; Xiao, Y.; Liang, P.; Wu, K. Influence of seasonal migration on evolution of insecticide resistance in Plutella xylostella. Insect Sci. 2022, 29, 496–504. [Google Scholar] [CrossRef]
  4. Christiaens, O.; Whyard, S.; Vélez, A.M.; Smagghe, G. Double-stranded RNA technology to control insect pests: Current status and challenges. Front. Plant Sci. 2020, 11, 451. [Google Scholar] [CrossRef]
  5. Liu, S.; Jaouannet, M.; Dempsey, D.A.; Imani, J.; Coustau, C.; Kogel, K.H. RNA-based technologies for insect control in plant production. Biotechnol. Adv. 2020, 39, 107463. [Google Scholar] [CrossRef]
  6. Zhu, K.Y.; Palli, S.R. Mechanisms, applications, and challenges of insect RNA interference. Annu. Rev. Entomol. 2020, 65, 293–311. [Google Scholar] [CrossRef]
  7. Shen, C.H.; Jin, L.; Fu, K.Y.; Guo, W.C.; Li, G.Q. RNA interference targeting Ras GTPase gene Ran causes larval and adult lethality in Leptinotarsa decemlineata. Pest Manag. Sci. 2022, 78, 3849–3858. [Google Scholar] [CrossRef]
  8. Zhang, J.; Khan, S.A.; Hasse, C.; Ruf, S.; Heckel, D.G.; Bock, R. Full crop protection from an insect pest by expression of long double-stranded RNAs in plastids. Science 2015, 347, 991–994. [Google Scholar] [CrossRef]
  9. Wu, M.; Dong, Y.; Zhang, Q.; Li, S.; Chang, L.; Loiacono, F.V.; Ruf, S.; Zhang, J.; Bock, R. Efficient control of western flower thrips by plastid-mediated RNA interference. Proc. Natl. Acad. Sci. USA 2022, 119, e2120081119. [Google Scholar] [CrossRef]
  10. Liu, F.; Yang, B.; Zhang, A.; Ding, D.; Wang, G. Plant-mediated RNAi for controlling Apolygus lucorum. Front. Plant. Sci. 2019, 10, 64. [Google Scholar] [CrossRef] [PubMed]
  11. Guan, R.B.; Li, H.C.; Miao, X.X. RNAi pest control and enhanced BT insecticidal efficiency achieved by dsRNA of chymotrypsin-like genes in Ostrinia furnacalis. J. Pest Sci. 2017, 90, 745–757. [Google Scholar] [CrossRef]
  12. Vatanparast, M.; Kazzazi, M.; Sajjadian, S.M.; Park, Y. Knockdown of Helicoverpa armigera protease genes affects its growth and mortality via RNA interference. Arch. Insect Biochem. Physiol. 2021, 108, e21840. [Google Scholar] [CrossRef] [PubMed]
  13. Jiang, L.H.; Mu, L.L.; Jin, L.; Anjum, A.A.; Li, G.Q. Silencing uridine diphosphate N-acetylglucosamine pyrophosphorylase gene impairs larval development in Henosepilachna vigintioctopunctata. Pest Manag. Sci. 2021, 78, 3894–3902. [Google Scholar]
  14. Liu, D.; Asad, M.; Liao, J.; Chen, J.; Li, J.; Chu, X.; Pang, S.; Tariq, M.; Abbas, A.N.; Yang, G. The potential role of the Piwi gene in the development and reproduction of Plutella xylostella. Int. J. Mol. Sci. 2023, 24, 12321. [Google Scholar] [CrossRef]
  15. Hou, Q.L.; Zhang, H.Q.; Zhu, J.N.; Chen, E.H. Tyrosine hydroxylase is required for the larval-pupal transformation and immunity of Plutella xylostella: Potential for pest management. J. Agric. Food Chem. 2024, 72, 27818–27829. [Google Scholar]
  16. Zeng, J.; Kang, W.N.; Jin, L.; Anjum, A.A.; Li, G.Q. Knockdown of Vacuolar ATPase Subunit G Gene affects larval survival and impaired pupation and adult emergence in Henosepilachna vigintioctopunctata. Insects 2021, 12, 935. [Google Scholar] [CrossRef]
  17. Zeng, J.; Mu, L.L.; Jin, L.; Ali Anjum, A.; Li, G.Q. RNAi of vacuolar-type H (+)-ATPase genes causes growth delay and molting defect in Henosepilachna vigintioctopunctata. Bull. Entomol. Res. 2021, 111, 705–714. [Google Scholar] [CrossRef]
  18. Zeng, J.; Kang, W.N.; Jin, L.; Anjum, A.A.; Li, G.Q. Vacuolar ATPase subunit F is critical for larval survival in Henosepilachna vigintioctopunctata. Insect Mol. Biol. 2022, 31, 177–189. [Google Scholar]
  19. Fu, K.Y.; Guo, W.C.; Lü, F.G.; Liu, X.P.; Li, G.Q. Response of the vacuolar ATPase subunit E to RNA interference and four chemical pesticides in Leptinotarsa decemlineata (Say). Pestic. Biochem. Physiol. 2014, 114, 16–23. [Google Scholar] [CrossRef]
  20. McGuire, C.; Stransky, L.; Cotter, K.; Forgac, M. Regulation of V-ATPase activity. Front. Biosci. Landmark Ed. 2017, 22, 609–622. [Google Scholar] [PubMed]
  21. Song, Q.; Meng, B.; Xu, H.; Mao, Z. The emerging roles of vacuolar-type ATPase-dependent Lysosomal acidification in neurodegenerative diseases. Transl. Neurodegener. 2020, 9, 17. [Google Scholar] [CrossRef] [PubMed]
  22. Abbas, Y.M.; Wu, D.; Bueler, S.A.; Robinson, C.V.; Rubinstein, J.L. Structure of V-ATPase from the mammalian brain. Science 2020, 367, 1240–1246. [Google Scholar] [CrossRef]
  23. Kitagawa, N.; Mazon, H.; Heck, A.J.R.; Wilkens, S. Stoichiometry of the peripheral stalk subunits E and G of yeast V1-ATPase determined by mass spectrometry. J. Biol. Chem. 2008, 283, 3329–3337. [Google Scholar] [CrossRef]
  24. Muench, S.P.; Huss, M.; Song, C.F.; Phillips, C.; Wieczorek, H.; Trinick, J.; Harrison, M.A. Cryo-electron microscopy of the vacuolar ATPase motor reveals its mechanical and regulatory complexity. J. Mol. Biol. 2009, 386, 989–999. [Google Scholar] [CrossRef]
  25. Mazhab-Jafari, M.T.; Rohou, A.; Schmidt, C.; Bueler, S.A.; Benlekbir, S.; Robinson, C.V.; Rubinstein, J.L. Atomic model for the membrane-embedded V(O) motor of a eukaryotic V-ATPase. Nature 2016, 539, 118–122. [Google Scholar] [CrossRef]
  26. Roh, S.H.; Stam, N.J.; Hryc, C.F.; Couoh-Cardel, S.; Pintilie, G.; Chiu, W.; Wilkens, S. The 3.5-Å CryoEM structure of nanodisc-reconstituted yeast vacuolar ATPase V(o) proton channel. Mol. Cell 2018, 69, 993–1004.e3. [Google Scholar] [CrossRef] [PubMed]
  27. Bonin, C.P.; Mann, R.S. A piggyBac transposon gene trap for the analysis of gene expression and function in drosophila. Genetics 2004, 167, 1801–1811. [Google Scholar] [CrossRef] [PubMed]
  28. Zeng, J.; Mu, L.L.; Jin, L.; Anjum, A.A.; Li, G.Q. Evaluation of three vacuolar ATPase genes as potential RNAi target in Henosepilachna vigintioctopunctata. J. Asia Pac. Entomol. 2021, 24, 55–63. [Google Scholar] [CrossRef]
  29. Xie, C.; Xiong, L.; Ye, M.; Shen, L.; Li, J.; Zhang, Z.; You, M.; You, S. Genome-wide analysis of V-ATPase genes in Plutella xylostella (L.) and the potential role of PxVHA-G1 in resistance to Bacillus thuringiensis Cry1Ac toxin. Int. J. Biol. Macromol. 2022, 194, 74–83. [Google Scholar] [CrossRef]
  30. Niu, R.C.; Meng, F.X.; Zeng, Q.H.; Wang, Y.J.; Liu, T.X.; Chu, D.; Zhang, S.Z. Comprehensive transcriptomic analyses of silk-associated genes and functional characterization of key silk fibroins in Plutella xylostella. Int. J. Mol. Sci. 2025, 26, 2842. [Google Scholar] [CrossRef]
  31. Ze, L.J.; Xu, P.; Kang, W.N.; Wu, J.J.; Jin, L.; Anjum, A.A.; Li, G.Q. Disruption of kynurenine pathway reveals physiological importance of tryptophan catabolism in Henosepilachna vigintioctopunctata. Amino Acids 2021, 53, 1091–1104. [Google Scholar] [CrossRef] [PubMed]
  32. Xu, P.; Ze, L.J.; Kang, W.N.; Wu, J.J.; Jin, L.; Anjum, A.A.; Li, G.Q. Functional divergence of white genes in Henosepilachna vigintioctopunctata revealed by RNA interference. Insect Mol. Biol. 2020, 29, 466–476. [Google Scholar] [CrossRef]
  33. Wu, J.J.; Cheng, M.D.; Ze, L.J.; Shen, C.H.; Jin, L.; Li, G.Q. Dissecting the isoform-specific roles of FTZ-F1 in the larval-larval and larval-pupal ecdyses in Henosepilachna vigintioctopunctata. Insects 2022, 13, 228. [Google Scholar] [CrossRef]
  34. Hameed, M.S.; Wang, Z.; Vasseur, L.; Yang, G. Molecular Characterization and the Function of Argonaute3 in RNAi Pathway of Plutella xylostella. Int. J. Mol. Sci. 2018, 19, 1249. [Google Scholar] [CrossRef] [PubMed]
  35. Bustin, S.A.; Benes, V.; Garson, J.A.; Hellemans, J.; Huggett, J.; Kubista, M.; Mueller, R.; Nolan, T.; Pfaffl, M.W.; Shipley, G.L.; et al. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 2009, 55, 611–622. [Google Scholar] [CrossRef]
  36. Huvenne, H.; Smagghe, G. Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: A review. J. Insect Physiol. 2010, 56, 227–235. [Google Scholar] [CrossRef]
  37. Baum, J.A.; Bogaert, T.; Clinton, W.; Heck, G.R.; Feldmann, P.; Ilagan, O.; Johnson, S.; Plaetinck, G.; Munyikwa, T.; Pleau, M.; et al. Control of coleopteran insect pests through RNA interference. Nat. Biotechnol. 2007, 25, 1322–1326. [Google Scholar] [CrossRef]
  38. Mao, Y.B.; Cai, W.J.; Wang, J.W.; Hong, G.J.; Tao, X.Y.; Wang, L.J.; Huang, Y.P.; Chen, X.Y. Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat. Biotechnol. 2007, 25, 1307–1313. [Google Scholar] [CrossRef]
  39. Wytinck, N.; Manchur, C.L.; Li, V.H.; Whyard, S.; Belmonte, M.F. dsRNA uptake in plant pests and pathogens: Insights into RNAi-based insect and fungal control technology. Plants 2020, 9, 1780. [Google Scholar] [CrossRef]
  40. Yan, S.; Ren, B.; Zeng, B.; Shen, J. Improving RNAi efficiency for pest control in crop species. Biotechniques 2020, 68, 283–290. [Google Scholar] [CrossRef] [PubMed]
  41. Zotti, M.; Dos Santos, E.A.; Cagliari, D.; Christiaens, O.; Taning, C.N.T.; Smagghe, G. RNA interference technology in crop protection against arthropod pests, pathogens and nematodes. Pest Manag. Sci. 2018, 74, 1239–1250. [Google Scholar] [CrossRef]
  42. Spit, J.; Philips, A.; Wynant, N.; Santos, D.; Plaetinck, G.; Vanden Broeck, J. Knockdown of nuclease activity in the gut enhances RNAi efficiency in the Colorado potato beetle, Leptinotarsa decemlineata, but not in the desert locust, Schistocerca gregaria. Insect Biochem. Mol. Biol. 2017, 81, 103–116. [Google Scholar] [CrossRef]
  43. Luo, J.; Liang, S.; Li, J.; Xu, Z.; Li, L.; Zhu, B.; Li, Z.; Lei, C.; Lindsey, K.; Chen, L.; et al. A transgenic strategy for controlling plant bugs (Adelphocoris suturalis) through expression of double-stranded RNA homologous to fatty acyl-coenzyme A reductase in cotton. New Phytol. 2017, 215, 1173–1185. [Google Scholar] [CrossRef]
  44. Xu, L.; Duan, X.; Lv, Y.; Zhang, X.; Nie, Z.; Xie, C.; Ni, Z.; Liang, R. Silencing of an aphid carboxylesterase gene by use of plant-mediated RNAi impairs Sitobion avenae tolerance of Phoxim insecticides. Transgenic Res. 2014, 23, 389–396. [Google Scholar] [CrossRef]
  45. Mao, J.; Zeng, F. Plant-mediated RNAi of a gap gene-enhanced tobacco tolerance against the Myzus persicae. Transgenic Res. 2014, 23, 145–152. [Google Scholar] [CrossRef]
  46. Gunning, P.W.; Ghoshdastider, U.; Whitaker, S.; Popp, D.; Robinson, R.C. The evolution of compositionally and functionally distinct actin filaments. J. Cell Sci. 2015, 128, 2009–2019. [Google Scholar] [CrossRef] [PubMed]
  47. San Miguel, K.; Scott, J.G. The next generation of insecticides: dsRNA is stable as a foliar-applied insecticide. Pest Manag. Sci. 2016, 72, 801–809. [Google Scholar] [CrossRef]
  48. Park, M.H. The post-translational synthesis of a polyamine-derived amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF5A). J. Biochem. 2006, 139, 161–169. [Google Scholar] [CrossRef] [PubMed]
  49. Christiaens, O.; Iga, M.; Velarde, R.A.; Rougé, P.; Smagghe, G. Halloween genes and nuclear receptors in ecdysteroid biosynthesis and signalling in the pea aphid. Insect Mol. Biol. 2010, 19, 187–200. [Google Scholar] [CrossRef]
  50. Wu, J.J.; Mu, L.L.; Kang, W.N.; Ze, L.J.; Shen, C.H.; Jin, L.; Anjum, A.A.; Li, G.Q. RNA interference targeting ecdysone receptor blocks the larval-pupal transition in Henosepilachna vigintioctopunctata. Insect Sci. 2021, 28, 419–429. [Google Scholar] [CrossRef] [PubMed]
  51. Xu, Q.Y.; Deng, P.; Zhang, Q.; Li, A.; Fu, K.Y.; Guo, W.C.; Li, G.Q. Ecdysone receptor isoforms play distinct roles in larval-pupal-adult transition in Leptinotarsa decemlineata. Insect Sci. 2020, 27, 487–499. [Google Scholar] [CrossRef]
  52. Abe, M.; Saito, M.; Tsukahara, A.; Shiokawa, S.; Ueno, K.; Shimamura, H.; Nagano, M.; Toshima, J.Y.; Toshima, J. Functional complementation reveals that 9 of the 13 human V-ATPase subunits can functionally substitute for their yeast orthologs. J. Biol. Chem. 2019, 294, 8273–8285. [Google Scholar] [CrossRef]
  53. Wieczorek, H.; Beyenbach, K.W.; Huss, M.; Vitavska, O. Vacuolar-type proton pumps in insect epithelia. J. Exp. Biol. 2009, 212, 1611–1619. [Google Scholar] [CrossRef] [PubMed]
  54. Muench, S.P.; Scheres, S.H.; Huss, M.; Phillips, C.; Vitavska, O.; Wieczorek, H.; Trinick, J.; Harrison, M.A. Subunit positioning and stator filament stiffness in regulation and power transmission in the V1 motor of the Manduca sexta V-ATPase. J. Mol. Biol. 2014, 426, 286–300. [Google Scholar] [CrossRef]
  55. Liu, X.J.; Liang, X.Y.; Guo, J.; Shi, X.K.; Merzendorfer, H.; Zhu, K.Y.; Zhang, J.Z. V-ATPase subunit a is required for survival and midgut development of Locusta migratoria. Insect Mol. Biol. 2022, 31, 60–72. [Google Scholar] [CrossRef]
  56. Ma, Y.F.; Zhao, Y.Q.; Zhou, Y.Y.; Feng, H.Y.; Gong, L.L.; Zhang, M.Q.; Hull, J.J.; Dewer, Y.; Roy, A.; Smagghe, G.; et al. Nanoparticle-delivered RNAi-based pesticide target screening for the rice pest white-backed planthopper and risk assessment for a natural predator. Sci. Total Environ. 2024, 926, 171286. [Google Scholar] [CrossRef]
  57. Guan, R.B.; Li, H.C.; Fan, Y.J.; Hu, S.R.; Christiaens, O.; Smagghe, G.; Miao, X.X. A nuclease specific to lepidopteran insects suppresses RNAi. J. Biol. Chem. 2018, 293, 6011–6021. [Google Scholar] [CrossRef] [PubMed]
  58. Shukla, J.N.; Kalsi, M.; Sethi, A.; Narva, K.E.; Fishilevich, E.; Singh, S.; Mogilicherla, K.; Palli, S.R. Reduced stability and intracellular transport of dsRNA contribute to poor RNAi response in lepidopteran insects. RNA Biol. 2016, 13, 656–669. [Google Scholar] [CrossRef]
  59. Wang, K.; Peng, Y.; Pu, J.; Fu, W.; Wang, J.; Han, Z. Variation in RNAi efficacy among insect species is attributable to dsRNA degradation in vivo. Insect Biochem. Mol. Biol. 2016, 77, 1–9. [Google Scholar] [CrossRef]
  60. Liu, J.; He, Q.; Lin, X.; Smagghe, G. Recent progress in nanoparticle-mediated RNA interference in insects: Unveiling new frontiers in pest control. J. Insect Physiol. 2025, 104884. [Google Scholar] [CrossRef] [PubMed]
  61. Sandal, S.; Singh, S.; Bansal, G.; Kaur, R.; Mogilicherla, K.; Pandher, S.; Roy, A.; Kaur, G.; Rathore, P.; Kalia, A. Nanoparticle-Shielded dsRNA Delivery for Enhancing RNAi Efficiency in Cotton Spotted Bollworm Earias vittella (Lepidoptera: Nolidae). Int. J. Mol. Sci. 2023, 24, 9161. [Google Scholar] [CrossRef] [PubMed]
  62. Jiang, L.; Wang, Q.; Kang, Z.H.; Wen, J.X.; Yang, Y.B.; Lu, X.J.; Guo, W.; Zhao, D. Novel Environmentally Friendly RNAi Biopesticides: Targeting V-ATPase in Holotrichia parallela Larvae Using Layered Double Hydroxide Nanocomplexes. J. Agric. Food Chem. 2024, 72, 11381–11391. [Google Scholar] [CrossRef] [PubMed]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Article Metrics

Citations

Article Access Statistics

Journal Statistics
Multiple requests from the same IP address are counted as one view.
Download PDF
Biological Characteristics and Bacterial Community of Invasive Pest Corythucha ciliata (Hemiptera: Tingidae)
Previous
A DNA Barcode Inventory of Austrian Dragonfly and Damselfly (Insecta: Odonata) Species
Next