Knockdown of vATPase subunit A impairs larval-pupal development in Plutella xylostella

Plutella xylostella is a destructive pest damaging the cruciferous plants worldwide. Transgenic plants expressing dsRNA can be widely deployed for pest control. However, highly effective lethal target genes are currently lacking. Here, we assessed the efficacy of RNA interference (RNAi) against P. xylostella through microinjection of in vitro synthesized dsRNA targeting PxvATPaseA . The full-length ORF of PxvATPaseA was 1848 bp, encoding 615 amino acids. PxvATPaseA was widely transcribed during different developmental stages from egg to adult. It was abundantly expressed in the foregut, hindgut, and Malpighian tubules, compared with other tissue types. Injecting 800 ng of ds PxvATPaseA into the fourth-instar larvae greatly reduced the corresponding mRNA level by 1.88-fold on day 2, causing 45.8% larval mortality, and lastly 54.2% treated larvae successfully pupated. At a higher dose of 1200 ng of ds PxvATPaseA , RNAi efficacy was significantly enhanced. The expression level of PxvATPaseA was reduced by 2.22-fold and 1.92-fold on day 2 and 3, respectively, leading to 79.2% larval lethality, and only 20.8% of depleted larvae pupated. Our results indicate that PxvATPaseA is an effective target for managing P. xylostella . These findings will facilitate the development of new insecticides and the cultivation of pest-resistant plants.

The diamondback moth, Plutella xylostella, represents one of the most economically devastating pests affecting cruciferous crops, causing an estimated annual loss of US $770 million in China and US $4–5 billion globally (Shehzad et al., 2023; Yin et al., 2025). Its biological characteristics enable rapid evolution of resistance, with documented tolerance to over 100 chemical pesticides (Shehzad et al., 2023). Therefore, developing novel control strategies for this pest is urgently needed.

RNA interference (RNAi) is a conserved cellular regulatory pathway that utilizes double-stranded RNA (double-stranded RNA, dsRNA) to mediate precise, sequence-dependent gene silencing, which has garnered significant attention as a promising approach for sustainable pest management (Zhang et al., 2015, 2017; Li et al., 2022a). Recent studies have shown transgenic plants expressing dsRNA can be effectively deployed for pest management, which was classified into two distinct categories: categories nuclear transformation (NT) lines and plastid transformation (PT) lines (Zhang et al., 2015, 2017; Li et al., 2022a). For example, applying transgenic plant Nicotiana tabacum expressing dsRNA of different genes to manage Helicoverpa armigera (Xiong et al., 2013; Mamta et al., 2016), Myzus persicae (Zhao et al., 2016; Rauf et al., 2019), Bemisia tabaci (Dong et al., 2020), Chilo partellus, P. xylostella and Maruca vitrata (Rana et al., 2020). Furthermore, numerous transgenic plants, including Arabidopsis thaliana (Bhatia and Bhattacharya, 2018; Eakteiman et al., 2018), Gossypium hirsutum (Han et al., 2017), Oryza sativa (Yang et al., 2021), Glycine max (Dubey et al., 2017; Meng et al., 2017), Zea mays (Baum et al., 2007), Solanum tuberosum (Hussain et al., 2019; Naqqash et al., 2020), Lactuca sativa (Ibrahim et al., 2017), Brassica napus (Deng et al., 2024) are generated to control pests. Notably, transgenic B. napus producing dsCHS1 significantly reduced larval weight and increased mortality in P. xylostella (Deng et al., 2024). These findings demonstrate the feasibility of using dsRNA-expressing transgenic plants to manage P. xylostella populations. Nonetheless, the identification of effective molecular targets capable of inducing high lethality in pests stands as a fundamental requirement for the successful deployment of this transgenic technology.

Vacuolar-type H + -ATPase (V-ATPase) holoenzyme represents an evolutionarily conserved multisubunit complex ubiquitous across eukaryotic organisms. Its core function involves the hydrolysis of adenosine triphosphate (ATP), resulting in the formation of adenosine diphosphate (ADP) and inorganic phosphate, while concurrently translocating protons across cellular membranes (Zeng et al., 2021a, b, 2022). In terms of architecture, the V-ATPase holoenzyme exhibits a bipartite structural organization consisting of the cytosolic V1 sector (encompassing eight subunits A to H) that drives ATP breakdown, and the membrane-embedded V0 sector (consisting of five subnits a 1 d 1 c 4-5 c’ 1 c” 1) that facilitates proton translocation across cellular membranes (Mazhab-Jafari et al., 2016; McGuire et al., 2017; Roh et al., 2018; Abbas et al., 2020; Song et al., 2020).

Previous research has identified vATPaseA as an attractive candidate target for RNAi-mediated pest control. Silencing vATPaseA in Hyphantria cunea impaired intestinal development and increased larval mortality (Wang et al., 2024). In Plagiodera versicolora, knockdown of vATPase-A resulted in high mortality rates in both larvae and adults (Li et al., 2022b). Similar lethal effects have been observed in Acalymma vittatum (Kaplanoglu et al., 2026), Manduca sexta (Burke et al., 2019), Bemisia tabaci (Thakur et al., 2014) and H. armigera (Sharif et al., 2022). Collectively, these studies suggest that vATPaseA may serve as an effective target for controlling P. xylostella via RNAi.

In this study, we investigated the biological function of vATPaseA by assessing the impacts of two doses of ds vATPaseA by microinjection in P. xylostella. Our findings identify PxvATPaseA as a potential to aid in the development of new insecticides and the cultivation of pest-resistant plants.

Insect rearing

P. xylostella was collected from Brassica napus in Haidong City, Qinghai Province, China and raised on Wuhan City, Hubei Province, China. The larvae of P. xylostella were fed on excised leaves of B. napus under precise experimental conditions (Temperature: 26 ± 1 °C; Relative humidity: 60% ± 5%; Photoperiod: 16-hour light:8-hour dark). The adults were supplied with a 10% honey solution for nutrition.

Molecular cloning

The putative PxvATPaseA sequence was required from publicly available transcriptome and genome datasets of P. xylostella (Xie et al., 2022; Niu et al., 2025). To ensure sequence correctness, we performed PCR using 2 × Phanta™ Master Mix (Vazyme, Nanjing, China), based on gene-specific primers (Supplementary Table S1, Supplementary Figure S1). The calculated molecular weight and isoelectric point were calculated by the Compute pI/MW tool (https://web.expasy.org/compute_pi/). The accession number of full-length cDNA sequence was ON108624 (GenBank). Phylogenetic construction was carried out using MEGA-5 software (https://sourceforge.net/projects/mega5/), applying the neighbor-joining method with 1,000 bootstrap replications.

DsRNA synthesis

Two cDNA fragments corresponding to PxvATPaseA and enhanced green fluorescent protein, GFP) were PCR-amplified via gene-specific primers (Supplementary Table S1). To minimize off-target effects, we performed BLASTN analysis of target sequences against the P. xylostella transcriptome database, excluding regions with ≥20 bp contiguous homology to non-target genes. dsRNA was generated in vitro using the MEGAscript T7 High Yield Transcription Kit (Ambion, Austin, TX, USA) following the manufacturer’s protocol. Purified dsRNA concentrations were quantified by agarose gel electrophoresis and spectrophotometry (Nanodrop 1000). Aliquots were preserved at -80 °C until experimental application.

DsRNA microinjection

DsRNA microinjection was performed as described previously (Yu et al., 2025, 2026). Briefly, newly ecdysed fourth-instar larvae were injected with either 800 ng or 1200 ng of ds PxvATPaseA. The individuals injected with ds GFP were used as negative controls. Each treatment group consisted of 8 larvae per replicate, with 6 biological replicates per dose. At 2 and 3 days post-injection, three replicates were processed for qRT-PCR analysis to measure the efficiency of RNA interference. The remaining three replicates were observed daily for three weeks to record survival rate, weight changes, pupation and adult emergence rate. The defect phenotypes were photographed through a stereoscope MZ62 (Mingmei, Guangzhou, China).

Real-time quantitative PCR

Total RNA was isolated from all experimental samples to analyze temporal transcription patterns (eight individuals pooled per sample) and tissue-specific expression profiles (30 tissues pooled per sample). For treatment group analysis, total RNA was extracted using TRIzol reagent (YiFeiXue Tech, Nanjing, China), with each of samples containing 8 pooled larvae and three biological replicates. qRT-PCR was performed to measure the relative abundance using previously described protocols (Wang et al., 2024). The housekeeping gene ribosomal protein RPL32 (PxRPL13) was selected as the internal control. Each qRT-PCR assay included three biological replicates and three technical replicates to ensure reproducibility. Relative mRNA transcripts were computed via the 2 -ΔΔCt method.

Data analysis

Statistical analyses were carried out using SPSS version 26.0 for Windows (IBM Corp., Chicago, IL, USA). Data are presented as mean ± standard error (SE), and significant differences among treatment groups were determined using the Tukey-Kramer multiple comparison test. Survival curves were analyzed using GraphPad Prism version 8.0 with the log-rank (Mantel-Cox) test (95% confidence interval).

Identification of PxvATPaseA

A putative full-length PxvATPaseA was obtained in P. xylostella by mining the transcriptome and genome data. The full-length ORF of PxvATPaseA was 1848 bp, encoding 615 amino acids with a predicted molecular weight of 68.12 kDa and theoretical pI of 5.12. Domain analysis showed that Px vATPaseA contained a conserved V-ATPase_V1_A domain (Figure 1A).

Phylogenetic analysis of vATPaseA from 19 insect species was built to assess the evolutionary relationships (Figure 1B).These sequences were from five lepidoptera: Ostrinia furnacalis, M. sexta, Cnaphalocrocis medinalis, Bombyx mori, and P. xylostella; three diptera: Aedes aegypti, Ceratitis capitata, and Bactrocera dorsalis; three hemiptera: Nilaparvata lugens, B. tabaci, and Acyrthosiphon pisum; three hymenoptera: Apis mellifera, Apis florea, and Bombus impatiens; and five coleoptera: Propylea japonica, Henosepilachna vigintioctopunctata, Diabrotica virgifera virgifera, P. versicolora, and Leptinotarsa decemlineata. The analysis included species from five insect orders: Lepidoptera, Diptera, Hemiptera, Hymenoptera and Coleoptera. The resulting unrooted tree revealed that Px vATPaseA clustered within the Lepidoptera subclade (Figure 1B).

Expression analysis of PxvATPaseA

qRT-PCR was employed to analyze the temporal expression profile of the PxvATPaseA. PxvATPaseA was broadly expressed across all developmental stages. PxvATPaseA mRNA expression was significantly elevated in the early stage of each larval stage (second-, third- and fourth-instar larvae), and low at day 3 of the fourth-instar larvae (Figure 2A).

The tissue-specific expression pattern of PxvATPaseA was systematically analyzed across all examined tissues, including the head, foregut, midgut, hindgut, hemolymph, Malpighian tubules, and epidermis. The results indicated that PxvATPaseA was constitutively expressed in various tissues, with the highest expression observed in the foregut, hindgut, and Malpighian tubules, followed by moderate levels in the head, hemolymph, and midgut, and the lowest expression in the epidermis (Figure 2B).

Impacts of injecting 800 ng ds PxvATPaseA into fourth-instar larvae

To assess the effects of PxvATPaseA -targeted RNAi in P. xylostella, we performed gene silencing using dsRNA microinjection. Microinjection of 800 ng ds PxvATPaseA into newly-molted fourth-instar larvae resulted in a significant 46.8% reduction in PxvATPaseA mRNA levels by day 2 post-injection, but no significant difference was observed on day 3 (Figure 3A).

Knockdown of PxvATPaseA significantly inhibited larval development, with fresh weights lowered by 22.6% and 18.5% on day 3 and 4, respectively (Figure 3B). RNAi-mediated silencing of PxvATPaseA resulted in high larval mortality, with approximately 41.7% and 45.8% of treated larvae dying on days 3 and 4 post-injection, respectively, and the mortality rate of larvae in the ds GFP group was 8.33% (Figure 3C). Silencing PxvATPaseA disrupted larval-pupal development, resulting in successful pupation of only 54.2% of treated larvae (Figure 3D). In contrast, knockdown of PxvATPaseA had no significant effect on adult emergence rates (Figure 3E). Larvae treated with ds PxvATPaseA exhibited small and misshapen body size, gradually darkening and lastly dying (Figures 3F vs. 3G).

Effects of injecting 1200 ng ds PxvATPaseA into fourth-instar larvae

Injection of 1200 ng ds PxvATPaseA greatly lowered the PxvATPaseA transcript by 55.0% and 47.9% on days 2 and 3, respectively, compared with ds GFP -injected controls (Figure 4A).

Knockdown of PxvATPaseA exerted a potent inhibitory effect on larval growth, resulting in significant reductions in fresh weight of 28.9%, 32.8%, and 33.8% on days 2, 3, and 4 post-injection, respectively (Figure 4B). RNAi of PxvATPaseA led to larval mortality, reaching 54.8%, 75.0% and 79.2% on day 2, 3 and 4, respectively (Figure 4C). Ultimately, only 20.8% of ds PxvATPaseA -treated larvae pupated (Figure 4D), though the adult emergence rate did not differ significantly between treatment and control groups (Figure 4E). PxvATPaseA -silenced larvae exhibited a complete failure to undergo pupal ecdysis, displaying stunted growth and abnormal body morphology, compared to ds GFP -injected controls. These PxvATPaseA -treated hypomorphs gradually became withered, dried and blackened, and eventually died (Figures 4F vs. 4G).

vATPase is a multi-subunit complex involved in various biological functions across living organisms (Zeng et al., 2021c). To date, the function of vATPaseA in P. xylostella has not been characterized. In this study, the sequence of vATPaseA in P. xylostella was identified from the transcriptome and genome data. RNAi-mediated silencing of PxvATPaseA via microinjection of two distinct dsRNA doses inhibited larval development, impaired pupae and ultimately caused high larval mortality. These results support that PxvATPaseA is an effective molecular target for sustainable management of P. xylostella larvae.

Domain analysis indicated the Px vATPaseA protein is highly conserved across species, containing a V-ATPase_V1_A domain (Figure 1). Additionally, a phylogenetic tree constructed from vATPaseA amino acid sequences of 19 insect species revealed that Px vATPaseA clusters within the Lepidoptera subclade (Figure 1). These results highlight the potential of targeting vATPaseA as an alternative RNAi-based strategy for managing P. xylostella.

PxvATPaseA was broadly expressed throughout all developmental stages, from embryo (egg) to adult. The mRNA level of PxvATPaseA was high in the early stage of each larval stage (second-, third- and fourth-instar larvae), and low at day 3 of fourth-instar larvae (Figure 2). Comparatively, peak vATPaseA expression in H. cunea occurs in third-instar larvae (Wang et al., 2024), whereas in P. versicolora, the highest expression is observed in first-instar larvae (Li et al., 2022b). The elevated mRNA levels of PxvATPaseA during larval stages suggest a critical role larval growth and development in P. xylostella.

Tissue-specific expression patterns indicated that PxvATPaseA was highly expressed in the foregut, hindgut and Malpighian tubules (Figure 2). Consistent with our findings, in H. vigintioctopunctata, the mRNA level of vATPaseA was highest in the Malpighian tubules (Guo et al., 2021), while in P. versicolora, vATPaseA was highly transcribed in the hindgut and Malpighian tubules (Li et al., 2022b). These similar expression profiles indicate that vATPaseA serves a vital function in toxin metabolism and waste excretion (Sharif et al., 2022).

Although studies have shown that RNAi targeting PxTH (Hou et al., 2024), PxCHS1 (Deng et al., 2024), PxvATPasea that has an important function inion homeostasis (Yu et al., 2025), PxvATPaseE that underscores a critical role in nutrient absorption (Yu et al., 2026) induced high mortality in P. xylostella, lepidopteran pests can evolve resistance to RNAi through various mechanisms, including dsRNA instability, restricted systemic spread, endosomal entrapment, and dysfunction of core RNAi machinery (Cooper et al., 2019). Double-stranded ribonucleases (dsRNases), present in insect hemolymph and gut fluid, are widely recognized as a key barrier to RNA interference (RNAi) effectiveness (Cooper et al., 2019). By targeting dsRNA molecules circulating in bodily fluids, these enzymes compromise dsRNA stability, thereby impeding RNAi performance. Inhibiting dsRNase activity in insects can mitigate intracellular dsRNA breakdown and significantly boost RNAi efficiency (Yu et al., 2025). In additions, Encapsulating double-stranded RNA (dsRNA) within protective nanomaterials serves as an innovative delivery strategy for RNA interference (RNAi) applications, effectively preserving the structural integrity of dsRNA molecules. When combined with targeted inhibition of double-stranded ribonucleases (dsRNases), this approach minimizes intracellular dsRNA degradation and significantly enhances RNAi efficacy. These nanocarrier complexes exhibit high transduction efficiency and low cytotoxicity, facilitating efficient cellular uptake of dsRNA while shielding it from nuclease-mediated degradation under fluctuating environmental conditions, such as variable temperatures and dynamic medium compositions (Yu et al., 2026).Therefore, identifying highly effective lethal target genes is essential for sustainable control of lepidopteran pests such as P. xylostella.

Our results demonstrated that microinjection of ds PxvATPaseA at two doses significantly suppressed PxvATPaseA mRNA levels, reduced larval fresh weight, and caused high mortality (Figures 3, 4). Consistent with previous studies, knockdown of vATPaseA in H. cunea (Wang et al., 2024), H. vigintioctopunctata (Guo et al., 2021) and P. versicoloraa (Li et al., 2022b) resulted in significant lethal effects. Furthermore, transgenic B. napus expressing hpPxCHS1 increased larval mortality (Deng et al., 2024), suggesting that PxvATPaseA could similarly serve as an effective target for developing genetically modified crops to control P. xylostella.