Functional Analysis of vATPase subunits A Reveals Its Feasibilty as a Target for Managing Plutella xylostella

1. Introduction

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 [1,2]. Its biological characteristics enable rapid evolution of resistance, with documented tolerance to over 100 chemical pesticides [1]. Therefore, developing novel control strategies for this pest is urgently needed.

Recent studies have shown transgenic plants expressing double-stranded RNA (dsRNA) via RNA interference (RNAi) can be effectively deployed for pest management, which was classified into two distinct categories: categories nuclear transformation (NT) lines and plastid transformation (PT) lines [3,4,5]. For example, applying transgenic plant Nicotiana tabacum expressing dsRNA of different genes to manage Helicoverpa armigera [6,7], Myzus persicae [8,9], Bemisia tabaci [10], Chilo partellus, P. xylostella and Maruca vitrata [11]. Furthermore, numerous transgenic plants, including Arabidopsis thaliana [12,13], Gossypium hirsutum [14], Oryza sativa [15], Glycine max [16,17], Zea mays [18], Solanum tuberosum [19,20], Lactuca sativa [21], Brassica napus [22] are generated to control pests. Notably, transgenic B. napus producing dsCHS1 significantly reduced larval weight and increased mortality in P. xylostella [22]. 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 [23,24,25]. 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 that facilitates proton translocation across cellular membranes [26,27,28,29,30].

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 [31]. In Plagiodera versicolora, knockdown of vATPase-A resulted in high mortality rates in both larvae and adults [32]. Similar lethal effects have been observed in Acalymma vittatum [33], Manduca sexta [34], Bemisia tabaci [35] and H. armigera [36]. 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 dsvATPaseA by microinjection in P. xylostella. Our findings identify PxvATPaseA as a potential target for developing dsRNA-expressing transgenic crops to control this pest.

2. Materials and Methods

2.1. Insect Rearing

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.

2.2. Molecular Cloning

The putative PxvATPaseA sequence was required from publicly available transcriptome and genome datasets of P. xylostella [37,38]. To ensure sequence correctness, we performed PCR using gene-specific primers (Table S1). The accession number of full-length cDNA sequence was ON108624. Phylogenetic construction was carried out using MEGA-5 software, applying the neighbor-joining method with 1,000 bootstrap replications.

2.3. DsRNA Synthesis

Two cDNA fragments corresponding to PxvATPaseA and enhanced green fluorescent protein, GFP) were PCR-amplified via gene-specific primers (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.

2.4. DsRNA Microinjection

DsRNA microinjection was performed as described previously [39,40]. Briefly, newly ecdysed fourth-instar larvae were injected with either 800 ng or 1200 ng of dsPxvATPaseA. The individuals injected with dsGFP 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.

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

Total RNA was isolated from all experimental samples to analyze temporal transcription patterns and tissue-specific expression profiles. 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 [31]. The housekeeping gene ribosomal protein RPL32 (PxRPL32) 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.

2.6. 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).

3. Results

3.1. Identification of PxvATPaseA

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 PxvATPaseA contained a conserved V-ATPase_V1_A domain (Figure 1A). Phylogenetic analysis of vATPaseA from 19 insect species revealed that PxvATPaseA clustered within the Lepidoptera subclade (Figure 1B).

3.2. Expression Analysis of 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). 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).

3.3. Impacts of Injecting 800 ng dsPxvATPaseA into Fourth-instar Larvae

Microinjection of 800 ng dsPxvATPaseA into newly-molted fourth-instar larvae resulted in a significant 1.88-fold 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 (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 dsPxvATPaseA exhibited small and misshapen body size, gradually darkening and lastly dying (Figure 4A vs. 4B).

3.4. Effects of Injecting 1200 ng dsPxvATPaseA into Fourth-instar Larvae

Injection of 1200 ng dsPxvATPaseA greatly lowered the PxvATPaseA transcript by 2.22-fold and 1.92-fold on days 2 and 3, respectively (Figure 5A). 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 5B). RNAi of PxvATPaseA led to larval mortality, reaching 54.8%, 75.0% and 79.2% on day 2, 3 and 4, respectively (Figure 5C). Ultimately, only 20.8% of dsPxvATPaseA-treated larvae pupated (Figure 5D), though the adult emergence rate did not differ significantly between treatment and control groups (Figure 5E). PxvATPaseA-silenced larvae exhibited a complete failure to undergo pupal ecdysis, displaying stunted growth and abnormal body morphology, compared to dsGFP-injected controls. These PxvATPaseA-treated hypomorphs gradually became withered, dried and blackened, and eventually died (Figure 4C vs. 4D).

4. Discussion

vATPase is a multi-subunit complex involved in various biological functions across living organisms [41]. 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 effectilve molecular target for sustainable management of P. xylostella larvae.

Domain analysis indicated the PxvATPaseA 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 PxvATPaseA 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 [31], whereas in P. versicolora, the highest expression is observed in first-instar larvae [32]. The elevated mRNA levels of PxvATPaseA during larval stages suggest a critical role larval growth and development in P. xylostella.

Tissue-speciffc 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 [42], while in P. versicolora, vATPaseA was highly transcribed in the hindgut and Malpighian tubules [32]. These similar expression profiles indicate that vATPaseA serves a vital function in toxin metabolism and waste excretion [36].

Although studies have shown that RNAi targeting PxTH [43], PxCHS1 [22], PxvATPasea [39], PxvATPaseE [40] 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 [44]. 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 dsPxvATPaseA at two doses significantly suppressed PxvATPaseA mRNA levels, reduced larval fresh weight, and caused high mortality (Figure 3 and Figure 4). Consistent with previous studies, knockdown of vATPaseA in H. cunea [31], H. vigintioctopunctata [42] and P. versicoloraa [32] resulted in significant lethal effects. Furthermore, transgenic B. napus expressing hpPxCHS1 increased larval mortality [22], suggesting that PxvATPaseA could similarly serve as an effective target for developing genetically modified crops to control P. xylostella.