Synergistically Better than One: Co-Application of Grasshopper-Derived +ssRNA Virus and Imidacloprid Induces Acute Toxicity in Locusta migratoria

Abstract

Entomopathogenic viruses offer an eco-friendly biological approach for pest control, but their relatively slow action often limits practical applications. Synergistic interactions between insect viruses and chemical pesticides can amplify their control efficacy, reduce insecticide use, and thus alleviate associated risks. Here, we evaluated the combined effects of the gomphocerinae permutotetra-like virus (GPV) and the neurotoxic insecticide imidacloprid against nymphs of Locusta migratoria. In toxicity tests, neither GPV nor imidacloprid alone caused mortality from acute toxicity after 12 h (<30%), but co-application led to marked acute synergistic toxicity, significantly increasing mortality to 87% within 12 h and 93% by 96 h. Importantly, histopathological examination revealed that the synergistic treatment caused severe midgut damage, such as disrupted or absent microvilli, extensive cellular debris in the gut lumen, cell detachment from the basal lamina, and apical displacement of nuclei. Furthermore, RNA-seq and biochemical analyses showed that the cotreatment aberrantly regulated key genes involved in peritrophic membrane integrity, substantially elevated immune responses, and disrupted energy homeostasis, which collectively led to death. These critical insights on the mechanisms underpinning the synergistic action of viral and traditional chemical agents underscore the potential of such integrated strategies to rapidly, effectively, and safely control pests.

1. Introduction

Grasshoppers (locusts) such as Locusta migratoria are among the most destructive pests worldwide, notorious for their gregarious, migratory swarms that devour vegetation across vast areas, causing extensive agricultural losses and posing a severe threat to global food security [1,2,3]. Furthermore, the recent massive outbreak of the desert locust (Schistocerca gregaria) has brought renewed attention to the resurgence of locust plagues and underscored the urgent need for effective, efficient control measures [4]. In many counties, current locust management, especially in emergency situations, primarily relies on the application of traditional chemical insecticides that exert neurotoxic effects on these pests [4,5]. However, the use of synthetic insecticides has been widely debated because of their long-term risks to the environment, biodiversity, livestock, and human health [6]. Notably, their frequent and extensive use leads to the development of resistance in the target insects and thus a significant reduction in the efficacy of the insecticide [7].

Given these challenges, environmentally friendly and sustainable biopesticides such as entomopathogenic fungi and viruses have gained increased attention and use for field control in recent years. Paranosema (Nosema) locustae and Metarhizium spp. and Aspergillus oryzae have been widely used to control locusts and grasshoppers [8]. Although these alternative biopesticides are considered ecologically safe and less likely to induce resistance in target pests, their relatively slow speed of killing limits their effectiveness when rapid responses are critical such as against locust plagues [9,10].

This limitation has been addressed by integrating them with chemical insecticides to enhance insecticidal activity while reducing chemical inputs, thus providing a realistic option to meet safer, more effective solutions to control locust plagues. Indeed, several efforts have demonstrated a synergy between insect viruses and chemical insecticides that promotes efficacy while lessening risks. For example, a synergistic interaction between baculoviruses (AcMNPV and SpliNPV) and low concentrations of synthetic insecticides such as azadirachtin, emamectin, and metaflumizone enhances insecticidal activity against Spodoptera littoralis [10].

Imidacloprid (N-{(2E)-1-[(6-Chloropyridin-3-yl)methyl]imidazolidin-2-ylidene}nitramide), a classical neonicotinoid insecticide, is commonly used worldwide due to its selective neurotoxic action on insects and its favorable safety profile for humans and other vertebrates. It acts on nicotinic acetylcholine receptors (nAChRs) in insect central nervous systems, thereby disrupting normal neurotransmission [11]. It is effective in controlling piercing–sucking pests and against certain heteropterans, coleopterans, and lepidopterans [2,3,12]. However, little is known about its effectiveness and impact on locust populations such as L. migratoria, due to its high cost and the fact that its primary target is piercing–sucking pests rather than locusts.

In our search for candidates to control grasshoppers, we focused on entomopathogenic viruses because they are host-specific and thus have minimal impact on the environment and non-target organisms [13]. Using a metagenomic approach, we discovered a broad diversity of DNA and RNA viruses in grasshoppers [14,15]. We isolated and identified a positive-sense single-stranded (+ssRNA) virus, gomphocerinae permutotetra-like virus (GPV, family Permutotetraviridae), from the grasshopper Dasyhippus barbipes [14,15]. Similarly to other insect viruses, GPV is slow to kill its insect host, but it can be vertically transmitted to the offspring, and it has sublethal effects that reduce insect biomass, shorten adult lifespan, and impair ovary development, significantly reducing fecundity [14,15].

In this work, we tested the environmentally friendly +ssRNA virus GPV and the neurotoxic insecticide imidacloprid for synergistic interactions that might enhance the toxicity of imidacloprid and reduce insecticide input. Firstly, GPV and imidacloprid were assayed for toxicity and control efficacy when co-applied to control nymphs of L. migratoria. Subsequently, we examined the histopathology of the midgut of treated nymphs to characterize tissue damage. Finally, RNA-seq was performed to elucidate the underlying synergistic mechanisms, which were further validated by qPCR and energy substrate measurements. Our insights into the mechanisms driving the synergistic interactions between viral agents and conventional chemical insecticides should facilitate the development of efficient, safer strategies to markedly improve the toxicity of viral agents for integrated pest management.

2. Materials and Methods

2.1. Insects, Virus, and Chemical Reagents

The locusts L. migratoria were obtained from a laboratory colony originating from the Key Biocontrol Laboratory for Locusts at the China Agricultural University, Beijing, China. Nymphs of L. migratoria that had never been exposed to insecticides were reared on fresh wheat seedlings in a controlled-climate room (30 ± 2 °C, 60 ± 10% RH, 16L:8D) following the method reported in Ref. [16]. Second-day third-instar nymphs were used for all experiments. Gomphocerinae permutotetra-like virus (GPV) was isolated from a population of Dasyhippus barbipes, and GPV purification was performed using sucrose density gradient ultracentrifugation as previously described [14,15]. GPV virions were identified using qPCR with forward primer 5′-TTACGCCCTCAGGTTATTCC-3′ and reverse primer 5′-CCTACACCAACTGCCATTCC-3′. Imidacloprid (95%) was purchased from Shanghai Yuanye Biotechnology Co., LTD (Shanghai, China). All other reagents, such as 4% v/v paraformaldehyde, ethanol, and paraffin, were purchased from Beijing Chemical Works (Beijing, China).

2.2. GPV and Imidacloprid Treatments of Nymphs

A total of 320 nymphs were randomly assigned to one of four treatments:

(1)

GPV only: Nymphs were intracoelomically injected with 200 μL sterilized PBS containing 2 × 10⁷ virions via the third and fourth abdominal segments. After 24 h, 2 μL acetone was applied to the pronotum.

(2)

Imidacloprid only: Nymphs were injected with 200 μL sterilized PBS. After 24 h, 2 µL imidacloprid (dissolved in 10 mg/L acetone) was applied to the pronotum.

(3)

GPV + imidacloprid: GPV-infected nymphs were incubated for 24 h, then received 2 µL imidacloprid (dissolved in 10 mg/L acetone) on the pronotum.

(4)

Controls: Nymphs were injected with 200 μL sterile PBS, and after 24 h, 2 µL acetone was placed on the pronotum.

Insects for each treatment were then placed in a mesh cage in the climate room. Dead nymphs were counted every 12 h for 96 h. Nymphs were collected after 12 h of exposure to imidacloprid for RNA-seq, histopathology, and energy substrate measurements. The experiments were conducted with a set of four replicates, each replicate containing 20 nymphs.

2.3. Histopathological Observations of Midgut

The midgut of nymphal locusts was dissected at 12 h post-treatment with imidacloprid or acetone for hematoxylin–eosin (H&E) staining. The experiment was conducted as previously described [17]. Briefly, excised midgut tissues were washed in Mordue’s locust saline, then fixed in 4% v/v paraformaldehyde, washed in 70% ethanol, and embedded in paraffin. Serial sections (4 µm thick) were mounted onto a glass slide with H&E solution and examined for histopathological changes using a microscope (Nikon, Tokyo, Japan).

2.4. RNA-Seq Analyses and qPCR Validation

Total RNA from five independent samples per treatment was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and RNA purity was assessed with a NanoPhotometer UV-Vis spectrophotometer (Implen, Westlake Village, CA, USA). For each sample, 3 μg RNA per sample was used to construct cDNA libraries using the NEBNext Ultra RNA Library Prep Kit (NEB, Ipswich, MA, USA) following the manufacturer’s protocol. Sequencing was performed using a PE150 strategy on an Illumina NovaSeq6000 platform (Biomics Biotech Co., Ltd., Beijing, China). Clean reads were mapped to the recently reported genome of L. migratoria [18] using TopHat v2.0.12 software. Genes with a log₂|Fold change| > 1 and a false discovery rate (FDR) < 0.01 were defined as differentially expressed genes (DEGs). Gene Ontology (GO) and KEGG pathway enrichment analyses of DEGs were carried out using the GOseq R package (v4.4.2) and KOBAS software (v3.0), respectively.

The total RNA samples used for RNA-seq were also used for qPCR to validate the RNA-seq results. cDNA was synthesized using the SuperReal PreMix Plus (SYBR Green) reagent kit (TianGen, Beijing, China). The CFX Connect Real-Time PCR Detection System (Bio-Rad, Beijing, China) was used for the qPCR with the primers in Table S1, with an initial denaturation at 95 °C for 30 s, then 39 cycles of 95 °C for 5 s and 60 °C for 30 s. EF1α was used as the reference gene [19].

2.5. Quantification of Total Proteins, Total Sugars, Triglycerides, and ATP

Total proteins, total sugars, triglycerides, and ATP in nymphs treated with GPV + imidacloprid complex were quantified as previously described [20], with five biological replicates per treatment. Briefly, for each assay, 0.1 g of treated nymphs was homogenized in 1 mL of the appropriate extraction buffer, centrifuged, and the supernatant collected for measuring absorbance of the solutions using a SpectraMax i3x (Molecular Devices LLC, San Jose, CA, USA). Total protein content was measured using the BCA Protein Quantification Kit (Abbkine Scientific Co., Ltd., Wuhan, China), measuring absorbance at 562 nm. Total sugar content was assessed using the Total Carbohydrate Content Assay Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), with absorbance measured at 540 nm. Standard curves for total protein and total sugar assays were generated using serial dilutions of respective standards (Figures S1 and S2). Triglyceride concentrations were determined with the CheKine Micro Triglyceride Assay Kit (Abbkine Scientific Co., Ltd., Wuhan, China), measuring absorbance at 420 nm. ATP content was measured using the ATP Content Assay Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) with absorbance read at 340 nm.

2.6. Statistical Analysis

Data analysis was performed in the IBM SPSS 27.0 software and GraphPad Prism 8.0. Comparisons between groups were performed using independent Student’s t-test (* p < 0.05; ** p < 0.01; *** p < 0.001). Survival analyses were performed using the Kaplan–Meier method.

3. Results

3.1. Combined Treatment with GPV and Imidacloprid Caused Rapid Acute Toxicity

In the survival assays, L. migratoria nymphs were treated with one of four different treatments over 96 h. The treatment with GPV + imidacloprid rapidly induced high nymph mortality indicative of acute toxicity; 87% of nymphs died within 12 h, and 92.11% within 96 h. In contrast, the other three treatments (GPV alone, imidacloprid alone, and PBS-acetone control) caused no acute toxicity; mortality rates were low (<30% by 96 h; Figure 1). The rapid, enhanced mortality provided by the combined treatment demonstrates a potent synergistic interaction between GPV and imidacloprid against L. migratoria nymphs.

3.2. Combined GPV and Imidacloprid Treatment Induced Synergistic Midgut Epithelial Damage

As illustrated in Figure 2, in the controls, midgut epithelial cells had typical intact, well-organized microvilli. Treatment with GPV alone or imidacloprid alone resulted in a slight reduction in microvilli density. In contrast, co-application of GPV and imidacloprid induced pronounced midgut damage with apoptotic features, including severely disrupted or absent microvilli in various places, extensive cellular debris in the gut lumen, cell detachment from the basal lamina, and apical displacement of nuclei.

3.3. GPV + Imidacloprid Treatment Induced More Transcriptional Alterations in Nymphs

To explore the molecular mechanisms underlying the acute toxicity of GPV + imidacloprid, we conducted transcriptomic analyses to evaluate changes in gene expression profile in nymphs following combined GPV and imidacloprid treatment. Compared to the imidacloprid treatment, GPV + imidacloprid resulted in 215 differentially expressed genes (DEGs) in nymphs, of which 89 were upregulated and 126 were downregulated (Figure 3A). GPV + imidacloprid considerably disrupted the physiological processes and metabolic homeostasis vital to nymph survival and development, including amino acid transport and metabolism (4 up-, 18 downregulated), carbohydrate transport and metabolism (5 up-, 13 downregulated), lipid transport and metabolism (3 up-, 8 downregulated), nucleotide transport and metabolism (2 downregulated) and replication, recombination, and repair (1 downregulated) (Figure 3B). Specifically, almost all (26 of 27) DEGs related to digestive enzymes were downregulated, including glucosidase involved in carbohydrate catabolism, as well as trypsin, chymotrypsin, elastase, carboxypeptidase, and aminopeptidase involved in proteolysis (Figure 4). In addition, GPV + imidacloprid significantly influenced the innate immunity pathway (10 upregulated), chitin degradation (4 upregulated), detoxification (8 up- and 15 downregulated), hippo signaling pathway (1 downregulated), insect hormone biosynthesis and metabolism (4 up-, 11 downregulated), and longevity regulating pathway (2 downregulated) (Figure 3 and Figure 4).

In contrast to the GPV + imidacloprid versus imidacloprid comparison group, the GPV + imidacloprid versus GPV comparison group had more upregulated DEGs (171) than downregulated DEGs (82) (Figure S3). Many of the induced DEGs were involved in carbohydrate transport and metabolism, insect hormone biosynthesis, and metabolism and detoxification enzymes. In the Venn diagram to illustrate shared and unique DEGs between treatments, 108 DEGs were shared across the two treatments (Figure 3C). Notably, GO and KEGG enrichment analyses showed that these shared DEGs were predominantly associated with amino acid transport and metabolism, carbohydrate transport and metabolism, lipid transport and metabolism and immune response (Figure 3D).

The qPCR analysis of 12 randomly selected genes to validate the RNA-seq results showed that their expression patterns were consistent with those shown by the RNA-seq data, confirming the reliability and reproducibility of the transcriptomic analysis (Figure 5).

3.4. Imidacloprid + GPV Treatment Significantly Impairs Nymph Energy Metabolism

After imidacloprid + GPV treatment, the DEGs associated with energy metabolism, including protein metabolism, sugar metabolism, and lipid metabolism, were predominantly downregulated. Therefore, to further assess the impact of imidacloprid + GPV treatment on nymph physiology, the contents of total proteins, total sugars, triglycerides and ATP were quantified. As shown in Figure 6, GPV + imidacloprid significantly reduced all measured energy reserves compared to imidacloprid alone. Specifically, total protein contents decreased from 6.04 to 5.03 mg/mL, total sugars from 17.34 to 14.14 mg/g, triglycerides from 63.34 to 39.27 mg/g, and ATP content from 701.09 to 179.47 μmol/mL. Collectively, these findings indicate that the combined treatment imposes a pronounced impact on the energy metabolism in the nymphs.

4. Discussion

Our recent metagenomic and virome surveys of grasshoppers collected from fields in China harbor a diverse array of DNA and RNA viruses, facilitating the identification and characterization of several novel pathogenic viruses including the +ssRNA virus GPV, thereby paving the way for the development of new biological control agents [14,15]. The GPV was found to be vertically transmitted from infected female locusts to their offspring, and its infection led to significant reductions in body weight and inhibited reproductive performance, including decreased ovarian weight and impaired ovarian development [15]. However, its slow-acting lethality will limit its widespread application as a microbial insecticide. Here, we demonstrated that GPV alone and imidacloprid alone did not cause acute toxicity in L. migratoria nymphs, whereas their co-application exerted acute toxicity, achieving 87% mortality within 12 h (Figure 1). This combined treatment resulted in synergistic interaction and offers an efficient and environmentally safe strategy for the integrated management of Orthoptera pest populations in China, achieving synergistic efficacy with reduced doses of imidacloprid; however, it should be noted that imidacloprid is restricted in some countries and regions, such as the EU, due to its ecotoxicological properties. Similarly, the co-application of Plutella xylostella granulovirus (PxGV) with a low dose of azadirachtin, or Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) with either emamectin benzoate or chlorfenapyr, and S. frugiperda multiple nucleopolyhedrovirus (SfMNPV) with spinetoram significantly enhance insecticidal efficacy compared to treatment with the individual components [7,21]. Furthermore, SeMNPV combined with phytochemicals such as genistein, kaempferol, quercitrin, and coumarin also exhibited synergistic effects against S. exigua larvae [22]. These findings underscore the potential of integrating viral agents with synthetic and natural insecticides for improving the efficacy and safety of pest management.

Consistent with the pronounced acute synergistic toxicity of GPV + imidacloprid, the nymphal midgut epithelium was much more damaged (Figure 2). Insect midgut serves as a crucial physiological barrier to exogenous substances, and its disruption could facilitate increased penetration of toxic imidacloprid, thereby enhancing insecticidal efficacy. Similarly to our findings, previous research has reported that midgut epithelial injury in Apis mellifera was greater after simultaneous exposure to the insecticide acetamiprid and the pathogen Nosema ceranae, thereby facilitating increased insecticide uptake and elevating larval mortality [23]. Also, Huang et al. (2025) [24] demonstrated that co-treatment of S. frugiperda larvae with zein nanoparticles and the fungicide polyoxin B causes more severe midgut tissue destruction than either agent alone, ultimately achieving excellent insecticidal effects. Collectively, these results suggest that impairment of the midgut tissues due to synergism between biological and chemical agents represents a common mechanism contributing to enhanced insecticidal activity. The peritrophic membrane is a key component of the insect midgut, primarily composed of chitin, cuticle protein, and peritrophins [24,25]. Peritrophins have been reported to emerge from the acquisition of chitin binding peritrophin A [26]. Our RNA-seq analysis demonstrated that the co-application of GPV and imidacloprid led to aberrant expression of some genes encoding chitin deacetylase (4 upregulated DEGs), chitin binding peritrophin A (4 up- and 1 downregulated DEGs), and cuticle protein (3 up- and 2 downregulated DEGs) (Figure 3). These results suggest that the combined treatment influences the transcriptional regulation of key genes involved in the formation and maintenance of the peritrophic membrane.

Compared to the transcriptome in nymphs treated with imidacloprid alone, GPV + imidacloprid resulted in the upregulation of all three DEGs encoding peptidoglycan recognition proteins (PGRPs), which participate in the recognition of the pathogenic microorganisms, thereby leading to the activation of the insect’s innate immune response [27]. Moreover, the expression of several key immune-related genes was significantly upregulated, including those encoding ankyrin (2 DEGs), peroxidase (1 DEG), serpin (1 DEG), and suppressor of cytokine signaling (SOCS; 1 DEG). Also upregulated were vacuolar sorting protein 9 (VPS9) and a CD36-related receptor, which are involved in the phagosome pathway. The phagosome pathway is involved in cellular uptake and degradation of pathogenic microorganisms and large particles [28] and thus plays a critical role in insect immune pathway. Members of the vacuolar protein sorting (VPS) family are involved in vesicle trafficking [29], and members of the CD36 family act as scavenger receptors for pathogens and as cofactors for Toll-like receptors by facilitating pathogen recognition [30]. The above findings therefore suggested that GPV + imidacloprid treatment more robustly activates immune responses in nymphs, enhancing their defense against invasion by the pathogenic microorganism GPV. However, it is well-established that sustained immune activation can adversely affect growth and development [31], which may partly account for the acute synergistic toxic effects after co-application of GPV and imidacloprid.

Notably, the combined application of GPV and imidacloprid also led to the upregulation of several important genes, including those that encode myo-inositol oxygenase (MIOX; 3 DEGs), C2H2 type zinc finger transcriptional factor (C2H2-ZF TF; 3 DEGs), BTB/POZ domain-containing protein (3 DEGs), and tyrosine aminotransferase (1 DEG). MIOX catalyzes the first step in the catabolism of myo-inositol and is crucial for regulating myo-inositol levels, which are essential for maintaining balanced metabolism, growth, and development [32]. The upregulation of MIOX, as observed in this study, could lead to decreased myo-inositol levels, potentially causing metabolic and neurodegenerative disorders. C2H2-ZF TFs and BTB/POZ domain-containing proteins play a critical role in various biological processes in insects, including growth and development, reproduction, and immune response [33,34]. For example, in L. migratoria, LmRn encodes the classical C2H2-ZF TF, which is essential for mediating pathways involved in growth and organ development [33].

Proteins, carbohydrates, and lipids, primarily triglycerides, serve as primary energy sources for insect growth and development [25,27]. Interestingly, GO and KEGG enrichment analyses highlighted significant downregulation of numerous DEGs associated with nutrient and energy metabolism (Figure 3 and Figure 4). Consistently, GPV + imidacloprid treatment markedly reduced levels of total proteins, sugars, triglycerides, and ATP in nymphs (Figure 6), confirming disruptions at both transcriptomic and biochemical levels. This collapse of all three macromolecular energy sources, together with sustained ATP reduction, likely arises from a neurotoxicity-induced feeding disruption, which limits energy intake and energy-demanding compensatory processes, which further accelerate substantial ATP consumption [20]. Ultimately, this persistent disruption of energy homeostasis exceeds physiological limits, killing the nymphs.

As shown by our RNA-seq and qPCR analyses, GPV + imidacloprid treatment also led to the downregulation of numerous key genes, such as all four UGT, all six hexamerin, and all two HSP genes (Figure 3 and Figure 5). UGTs catalyze the conjugation of UDP-glucose with small lipophilic xenobiotic compounds, including insecticides and plant secondary metabolites, facilitating their elimination and excretion [25,35]. Hexamerins have been shown to play multifaceted roles, participating in hormone transport, immune defense, metamorphosis, and phase transition from the solitary to the gregarious lifestyle in L. migratoria and binding to lipophilic insecticides [36,37]. HSP genes play critical roles not only in mitigating detrimental effects of various stresses, but also in modulating insect reproduction and longevity [35,38], In our study, similarly, one downregulated HSP gene (g145474) was annotated for the longevity regulating pathway. Also downregulated was another gene (g42490) involved in the longevity regulating pathway, which is annotated as the stearoyl-CoA desaturase gene. Previous research has shown that silencing the stearoyl-CoA desaturase gene in mosquitoes results in a highly skewed ratio of SFA to UFA, compromised integrity of the midgut epithelial cells, and acute auto-inflammation, ultimately significantly affecting the survival and reproductive success [39].

All seven genes involved in juvenile hormone biosynthesis were also downregulated by the synergistic action of GPV and imidacloprid. Juvenile hormone (JH), a critical regulator in insects, is essential for metamorphosis and female reproductive maturation [40]. JH is synthesized in a gland beneath the brain of insects and is protected from degradation and transported to target tissues and cells by juvenile hormone binding proteins (JHBPs) [41]. Importantly, one JHBP gene was also downregulated after treatment with GPV and imidacloprid. These data indicate impaired JH synthesis and transport. The molting hormone (ecdysteroid) might also be influenced by the combination treatment. Regulation of this steroid hormone, primarily through its synthesis and inactivation, is essential for mediating developmental transitions in insects [42]. We found that one ecdysone oxidase gene was downregulated and four out of six ecdysteroid kinase-like family genes were upregulated, both known to play a critical role in the metabolism of ecdysteroids. Ecdysone oxidase catalyzes the oxidation of ecdysteroids into 3-dehydroecdysteroid [42], and ecdysteroid kinase-like family mediates reversible phosphorylation of the ecdysteroid, thus modulating the titers of active hormone [43,44]. Members of the SV2 family are essential for neuromodulation and neurotransmission, and dysfunction of SV2 can lead to impaired neurotransmission and various neurological disorders [20]. For example, knockout of BmSV2B in Bombyx mori led to developmental larval defects and faster death [45]. In our study, the downregulation of SV2 after GPV + imidacloprid treatment indicates that GPV intensified imidacloprid-induced neurotoxicity. Also downregulated after treatment with GPV and imidacloprid were six of eight trehalose transporters, which are crucial for stress adaptation and for moving trehalose disaccharide from the fat body to the hemolymph [39]. Silencing the trehalose transporter gene AgTreT1 in Anopheles gambiae reduces trehalose levels and decreases survival under stress [46].

5. Conclusions

In summary, our results demonstrated that co-application of the entomopathogenic virus GPV and the synthetic imidacloprid induced acute synergistic toxicity against L. migratoria nymphs, substantially surpassing the efficacy of either agent alone. This enhanced lethality is associated with multifaceted molecular and physiological disruptions, including severe histopathological damage to the midgut epithelium, aberrant regulation of peritrophic membrane integrity genes, hyperactivation of immune responses, and profound impairment of energy homeostasis, as evidenced by transcriptomic and biochemical analyses. Specifically, GPV amplified insecticide-induced toxicity and disrupted nutrient and energy metabolism, while accelerating midgut tissue degradation, collectively contributing to accelerated and higher insect mortality. These findings help elucidate the mechanistic basis for virus–insecticide synergy but also offer a promising strategy for rapid, sustainable locust control through the integration of eco-friendly viral agents with traditional chemical insecticides.