Construction of a nontoxic nano-pesticide and its co-application with natural predators for perfect cooperative pest management: an innovative strategy for pesticide reduction

Abstract

Historically, the widespread and unscientific application of chemical pesticides has posed serious threats to the ecosystem and human health. Hence, there is an urgent need for efficient alternative technologies for green pest management. In this context, the current study developed a nontoxic nano-pesticide and co-applied with natural predators with enhanced bioactivity toward insect pests, and co-applied the nano-pesticide and predator to propose an innovative pest control technique. The bio-toxicity of the star cationic polymer (SPc) for use in the nano-pesticide preparation was first evaluated with widely applied predatory stinkbug Arma custos. The SPc exhibited negligible toxicity to both the eggs and nymphs of the predators; however, its extremely high concentration led to nymph death, with a lethal concentration 50 (LC₅₀) value of 14.75 mg mL⁻¹via oral feeding. This SPc at an extremely high concentration primarily induced a considerable stress response in the predator, which augmented the processes of phagocytosis, exocytosis, and energy synthesis and ultimately led to its death. Subsequently, we developed a self-assembled tetraniliprole (TTP)/SPc complex via hydrogen bonding and van der Waals interactions, which disrupted the self-aggregated structure of TTP and reduced its particle size by nearly 10 fold. Impressively, the contact and stomach toxicity of SPc-loaded TTP were significantly improved against common cutworm Spodoptera litura, with corrected mortalities increasing by approximately 30%. Importantly, the predators displayed a strong predation selectivity for alive pests, while the application of the nano-pesticide did not show any negative influences on the predators. Thus, the co-application of the nano-pesticide and predatory stinkbug could achieve efficient pest control. In summary, a nontoxic nano-pesticide was successfully developed to be co-applied with a predator, which not only represents an innovative technology solution for green pest management but also would be beneficial for reducing the use of pesticides to minimize their adverse impacts on the environment.

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Environmental significance

The indiscriminate and frequent application of chemical pesticides has led to pesticide residue exceeding the recommended safety levels, which poses a significant obstacle to the progress of sustainable agriculture. Consequently, there has been a growing demand for environmentally friendly pest control techniques in recent years. Natural predators have emerged as popular choices for sustainable pest management. However, their efficient application faces pressing challenges in actual production. Nano-pesticides are drawing considerable attention due to their superior physical and chemical properties. The co-application of nano-pesticides with predatory insects may present a more favorable option for the sustainable management of harmful organisms. In this context, the current study developed a nano-pesticide that was nontoxic toward natural predators with enhanced bioactivity toward insect pests and proposes co-applying the nano-pesticide with a natural predator to as an innovative pest control technique. The proposed solution is also beneficial for reducing the use of pesticides to minimize their adverse impacts on the environment.

Introduction

Chemical pesticides have played a pivotal role in the rapid development of modern agriculture, contributing to increased crop yields and quality.^1,2 However, their indiscriminate and frequent application has led to pesticide residue exceeding the recommended safety levels and the fast development of resistance in insect pests, which poses a significant obstacle to the progress of sustainable agriculture.^3,4 Consequently, there has been a growing demand for environmentally friendly pest control techniques in recent years,^5,6 including green methods such as biological control, precise application of chemicals, and ecological regulation. These approaches can not only reduce the reliance on chemical pesticides but also contribute to maintaining a healthy and stable ecological environment.^7–10 Among these methods, natural predators and nano-pesticides have emerged as popular choices for sustainable pest management.

The application of biocontrol techniques aim to maintain the pest population below the economic threshold, highlighting good sustainability and environmental compatibility.¹¹ Within the realm of biocontrol, predatory insects have emerged as one pivotal research area.¹² Predatory insects such as parasitic wasps and ladybugs have been widely adopted for the sustainable management of aphids and certain lepidopteran pests.^13,14 They have gradually emerged as robust representatives of eco-friendly alternatives for pesticide reduction.⁸ However, the efficient application of predatory insects faces pressing challenges in actual production. For instance, their effectiveness tends to be relatively low when dealing with a high pest population density or complicated environments, which constrains the large-scale application of predatory insects.¹⁵

Research on nano-pesticides is developing rapidly in the agricultural field, drawing considerable attentions due to their superior physical and chemical properties.^16–18 Compared to traditional chemical pesticides, nano-pesticides exhibit enhanced dispersion, efficient delivery capabilities, and superior pest control effects, thereby reducing the application of pesticides and minimizing adverse impacts on the environmental and human health.^17,19–21 Our group designed and synthesized a star cationic polymer (SPc) that can assemble with various pesticides to reduce their particle size, increase their dispersibility, and improve their plant uptake, thus increasing the biological activity. The SPc is positively charged (+20.9 mV) with a particle size of 100.5 nm and molecular weight of about 40 296 Da.^22–24 For example, SPc can be used to load thiamethoxam or avermectin to prepare nano-pesticides, which increases the contact and stomach toxicity of the pesticides against aphids.^23,25 The co-application of nano-pesticides with predatory insects may present a more favorable option for the sustainable management of harmful organisms. Recent research demonstrated that the co-application of predatory ladybugs with nano-pesticides could overcome the slow-acting property of natural enemies for efficient aphid control.²⁶

There are several scientific and technical issues requiring thorough assessment before the co-application of nano-pesticides and predatory insects. First, systematic evaluation is necessary to determine whether nano-materials exhibit toxicity to predatory insects. Previous studies have revealed that nano-silicon dioxide on leaf and stem surfaces may be absorbed by the lipid layer, leading to the death of predatory insects.^27,28 Second, it is crucial to assess whether nano-pesticides can control pests effectively while showing nontoxic to predatory insects. Some studies have suggested that the nano-pesticides may display increased toxicity against predatory insects.^24,29 Lastly, the reduction amount of pesticides possible via the co-application of nano-pesticides and predatory insects also requires calculation. Tetraniliprole (TTP) is a bisamide insecticide, primarily targeting insect ryanodine receptors and exhibiting high insecticidal activity against lepidopteran pests.^30,31 The predatory stinkbug Arma custos (Hemiptera: pentatomidae) is a broad-spectrum predator that is widely distributed across numerous provinces in China.³² It possesses excellent control capability against various agricultural and forestry pests in the orders of Lepidoptera, Hemiptera, and Coleoptera.³³

The current study aimed to develop a nontoxic TTP/SPc complex (nano-pesticide) toward A. custos, and to co-apply this nano-pesticide with a predator for perfect cooperative pest management. The biotoxicity of SPc was first evaluated against the eggs and nymphs of predatory stinkbugs using immersion and feeding methods, and the lethal mechanism of SPc at high concentration was explored using transcriptomic analysis. Subsequently, the nano-pesticide was prepared by the incubation of TTP with SPc, and the self-assembly mechanism and characteristics of the TTP/SPc complex were elucidated by isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and transmission electron microscopy (TEM). Furthermore, the contact and stomach toxicity of the TTP/SPc complex were examined against a globally polyphagous pest common cutworm Spodoptera litura,³⁴ whose larvae can cause significant damage to crops under severe infestation, leading to substantial crop losses or even complete failure.^35,36 Finally, the predation selectivity of predatory stinkbugs was investigated, and the toxicity of the nano-pesticide was assessed against the predatory stinkbugs. The co-application of the nano-pesticide and predatory stinkbugs was employed to control the common cutworm. Overall, the current study developed a nano-pesticide that was nontoxic to predatory stinkbugs and could be co-applied with the predator for perfect cooperative pest management.

Materials and methods

Insects and chemicals

The predatory stinkbugs A. custos were fed with the larvae of Galleria mellonella bought from Tianjin Huiyude Biotechnology Co. (China). The larvae of the common cutworm S. litura were fed on an artificial diet purchased from Henan Jiyuan Baiyun Industrial Co. (China). All the insects were maintained under the controlled conditions of 26 °C, 50–60% relative humidity, and 14 hours light : 10 hours dark (14H : 10L) photoperiods.

Pure TTP (≥90%) was obtained from Shanghai Yuanye BioTechnology Co. (China). The raw materials for SPc synthesis included N,N,N′,N′,N′′-pentamethyl diethylenetriamine (PMDETA, 98%) and CuBr purchased from Sigma-Aldrich (USA), 2-(dimethylamino) ethyl methacrylate (DMAEMA) purchased from Energy Chemical (China), and 2-bromo-2-methylpropionyl bromide and tetrahydrofuran (THF) purchased from Heowns BioChem Technologies (China). All other reagents were bought from Beijing Chemical Works (China). The SPc was synthesized in a two-step process following the methods described by Li et al.²² and Jiang et al.³⁷ In the initial step, a solution containing pentaerythritol in dry THF and triethylamine at 0 °C was subjected to the gradual addition of 2-bromo-2-methylpropionyl bromide. After stirring for 24 h, the reaction was terminated with methanol. The ensuing residue was purified by recrystallization in cold ether, leading to the formation of the star initiator Pt–Br upon removal of the solvent. In the subsequent step, a flask equipped with a magnetic stirrer was charged with Pt–Br, DMAEMA, and dry THF. The mixture underwent nitrogen degassing for 30 min, followed by the addition of CuBr and PMDETA. Polymerization was conducted at 60 °C for 7 h. The reaction was quenched by cooling and air exposure, resulting in the final SPc product obtained in the form of a white powder. The structure of SPc was also confirmed by ¹H NMR (CDCl₃, Bruker 400) (Fig. S1†).

Biotoxicity assay of SPc against the predatory stinkbug

The biotoxicity of SPc was examined against the eggs and nymphs of the predatory stinkbug, which is the primary natural predator of S. litura. For the egg assay, three-day old eggs were individually immersed in SPc solutions (56.71, 30, and 10 mg mL⁻¹) for 30 s. Newly-hatched nymphs were removed every 12 h, and the hatching rates were calculated at 9 d after the immersion. Double-distilled water (ddH₂O) was employed as the control. Each treatment consisted of 30 eggs, and was repeated 4 times. For the nymph assay, the contact and stomach toxicity of SPc were both tested. The newly-hatched nymphs were immersed in SPc solutions (56.71, 40, 30, 20, and 10 mg mL⁻¹) for 20 s. Each treatment contained 20 nymphs, and was repeated 4 times. Additionally, the 2nd instar nymphs were fed with sterile cotton balls immersed in SPc solutions (40, 30, 20, 15, and 10 mg mL⁻¹) for 20 s. Each treatment contained 20 nymphs, and was repeated 3 times. The corrected survival rate was calculated at 4 d after the treatment. The dose–response data were analyzed to determine the lethal concentration 50 (LC₅₀) using POLOPlus 2.0 (LeOra Software, USA).

RNA-seq analysis of the lethal mechanism induced by SPc

The 2nd instar nymphs were fed with SPc at the concentration of 14.75 mg mL⁻¹ (LC₅₀) for 2 d. All the surviving nymphs were homogenized, and total RNA was extracted using the RNA Simple Total RNA kit (Tiangen Biotech Co., China). Again, ddH₂O was adopted as the control. Three independent samples were prepared for each treatment.

The transcript libraries were constructed via the Illumina HiSeq sequencing platform. Clean reads were obtained by removing the reads containing adapters or poly-N sequences, as well as low-quality reads with a quality score below Q10. These clean reads were further assembled using Trinity, and then the assembled transcripts were clustered to eliminate redundancy with Tgicl to obtain the Unigenes.³⁸ Bowtie2 was employed to align the clean reads to the reference gene sequences, and RSEM was used to calculate the transcript expression levels.^39,40 The Unigenes were annotated through BLASTX analysis against the GO and KEGG databases. The expression level of each transcript was quantified using the fragments per kilobase of transcript per million (FPKM) value. Differentially expressed gene (DEG) analysis was performed using DESeq2, with the screening criteria set at a fold change of ≥2.0 and a false discovery rate (FDR) of <0.05.^41,42

To validate the expression levels of the target genes, the quantitative real time polymerase chain reaction (qRT-PCR) was carried out. Total RNA was reverse transcribed using the HiScript III 1st Strand cDNA Synthesis kit (Vazyme, China) to obtain cDNA. All the primers are listed in Table S1.† qRT-PCR was carried out on an ABI QuantStudio 6 Fiex system (Thermo Fisher, USA) using the TransStart Top Green qPCR SuperMix (TransGen Biotech, China). The expression level of each target gene was normalized to the expression of the reference gene Beta-actin (gene ID: 31521) using the 2^−ΔΔCT method.⁴³

Loading capacity measurement

The loading capacity of SPc toward TTP was measured using the freeze-drying method.³⁷ First, TTP was dissolved in dimethyl sulfoxide (DMSO) and ddH₂O (6 : 14 v : v). The SPc solution (100 mg) was mixed with the TTP solution (100 mg). The mixture was then dialyzed using the regenerated cellulose with a molecular weight cutoff of 2000 Da (Shanghai Yuanye BioTechnology Co., China) for 12 h. During the dialysis, the solution outside the dialysis bag was changed every 4 h. The samples were freeze-dried utilizing a lyophilizer (Beijing Songyuanhuaxing Technology Development Co., China) and weighed. The pesticide loading content (PLC) was determined using the formula: PLC (%) = weight of insecticide loaded in complex ÷ weight of insecticide loaded complex × 100%.

Isothermal titration calorimetry (ITC) assay

The binding force between TTP and SPc was investigated to illustrate the self-assembly mechanism using a highly precise isothermal titration calorimetry (ITC) method. In this study, 200 μL solution of TTP (0.05 mmol L⁻¹) was titrated with 38 μL of SPc solution (0.5 mmol L⁻¹) using ITC200 (GE Co., USA). The heats of interaction during each injection were calculated by integrating each titration peak using Origin7 software (OriginLab Co., USA). The test temperature was 25 °C, and ΔG was calculated using the formula of ΔG = ΔH − TΔS.

Complex morphology characterization and particle-size measurement

The morphology characterizations of TTP (400 μg mL⁻¹) and TTP/SPc complex (mass ratio of 1 : 1) were performed by transmission electron microscopy (TEM, JEOL-1200, Japan). To prepare the samples for TEM analysis, the samples were dropped on to copper mesh with a carbon film (Beijing Zhongjing Technology Co., China). After natural drying, the samples were treated with 1% phosphotungstic acid and dried again before observation. The particle sizes of the above samples were measured using a particle sizer and zeta potential analyzer (Brookhaven NanoBrook Omni, USA) at 25 °C. Each treatment included 3 independent samples.

Contact and stomach toxicity assay of TTP/SPc complex against the common cutworm

The toxicity of TTP/SPc complex (mass ratio of 1 : 2.11 as determined by PLC) was assessed toward the 2nd instar larvae of the common cutworm. This mass ratio was subsequently adopted in the following experiments. For the contact toxicity assay, the larvae were directly immersed in TTP formulations (10, 5, and 1 mg L⁻¹), as well as the corresponding formulations of TTP/SPc complex for 20 s. For the stomach toxicity assay, the larvae were fed with artificial diets, which were immersed in the aforementioned formulations for 20 s. Again, ddH₂O was used as the control. The number of dead larvae was recorded at 2 d after the treatment, and the corrected mortality was calculated using the formula: corrected mortality = (mortality in treatment − mortality in control) ÷ (1 − mortality in control) × 100%. Each treatment included 20 larvae and was repeated 3 times. The concentration–mortality data were analyzed to obtain the lethal concentration 50 (LC₅₀) using POLOPlus 2.0 (LeOra Software, USA).

Predation selectivity assay of the predatory stinkbug toward alive and dead common cutworm

To determine whether the nymphs of the predatory stinkbugs prefer to consume alive or dead larvae of the common cutworm, the 5 second instar nymphs starved for 6 h were fed with 10 alive and 10 dead larvae. The predation preference of the nymphs was recorded at 24 h after the treatment. The experiment was repeated 4 times.

Bioactivity assay of TTP/SPc complex toward the predatory stinkbug

The bioactivity of TTP/SPc complex was assessed toward newly-hatched nymphs via both immersion and oral feeding methods. The nymphs were immersed in the TTP formulation (5 mg L⁻¹) and corresponding formulation of TTP/SPc complex for 20 s, respectively. Simultaneously, sterile cotton balls immersed in the above solutions for 20 s were used to feed nymphs. The survival rate was recorded at 2 d after the treatment. Each treatment included 20 nymphs and was repeated 4 times.

Co-application of the TTP/SPc complex and predatory stinkbug to control the common cutworm

Three treatments were employed to evaluate the control effects: (1) 20 second instar nymphs of predatory stinkbugs were applied to control 40 second instar larvae of common cutworms, (2) 40 second instar larvae of common cutworms were immersed in TTP/SPc complex solution (5 mg L⁻¹) for 20 s, (3) predatory stinkbugs, common cutworms, and artificial diets were immersed in TTP/SPc complex solution (5 mg L⁻¹) for 20 s, and placed in a box. The mortality of the common cutworms was recorded at 2 d after the treatment. Each treatment was repeated 4 times.

Data analysis

The statistical analysis was carried out using SPSS 27.0.1 software (SPSS Inc., USA). The descriptive statistics are presented as the mean value and standard errors of the mean. The Tukey HSD test or independent t-test was used to analyze the data, with P = 0.05 indicating significance.

Results

Negligible biotoxicity of SPc toward the predatory stinkbug

In the current study, a slight reduction in the hatching rate of the predatory stinkbug was observed after SPc treatment (Fig. 1A). The greatest decline was at the concentration of 56.71 mg mL⁻¹ with a hatching rate of 84%. Nevertheless, it did not exhibit significant difference compared to the control. Similarly, nearly no death of newly-hatched nymphs was observed after the immersion in SPc formulations, suggesting its negligible contact toxicity toward nymphs (Fig. 1B). However, the SPc exhibited stomach toxicity at 4 d after oral feeding, with an LC₅₀ value of 14.75 mg mL⁻¹ (Fig. 1C and Table 1).

Fig. 1 Biotoxicity of SPc against predatory stinkbug. (A) Hatching rate of eggs immersed in SPc solution, with ddH₂O used as the control. Each treatment included 30 eggs, and was repeated 4 times. (B) Contact toxicity of SPc against the newly-hatched nymphs. Nymphs were immersed in SPc solutions, and the corrected survival rate was calculated at 4 d after the treatment. Each treatment contained 20 nymphs, and was repeated 4 times. (C) Stomach toxicity of SPc against the 2nd instar nymphs. Nymphs were fed with sterile cotton balls immersed in SPc solutions. Each treatment contained 20 nymphs, and was repeated 3 times. Different letters indicate significant differences according to the Tukey HSD test at the P = 0.05 level of significance.

Table 1 Toxicity of SPc toward the 2nd instar nymphs of Arma custos using the oral feeding method

LC30 (mg mL^−1) (95% confidence limits)	LC50 (mg mL^−1) (95% confidence limits)	Slope ± SE	χ ^2 (df)	P value
9.606 (7.173–11.551)	14.747 (12.437–16.805)	2.817 ± 0.389	4.530 (13)	0.984

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Lethal mechanism of SPc toward the predatory stinkbug

To explore the potential mechanism of SPc-induced death, the total RNA was extracted from the surviving nymphs fed with SPc (14.75 mg mL⁻¹) for RNA-seq analysis. The sequencing quality was high enough for further analysis (Table S2†). Compared with the control, the expression of 20 604 genes was obviously altered after the oral feeding with SPc. A total of 11 305 genes were upregulated, and 9299 genes were downregulated (Fig. 2A). The DEGs could be divided into three categories: biological process, cellular component, and molecular function (Fig. 2C). The DEGs were mainly enriched in the pathways for the lipid metabolic process, metabolic process, extracellular region, structural constituent of cuticle, chitin binding, and heme binding, etc.

Fig. 2 RNA-seq analysis to explore the potential mechanism of SPc-induced death. (A) Analysis of the differentially expressed genes (DEGs) with a volcano plot. Up- and down-regulated genes are represented by red and blue dots, respectively. (B) Heat maps of the transport-related and energy-related genes. Highly expressed genes are shown in red, whereas genes with low expression levels are shown in blue. (C) GO enrichment analysis of the DEGs. Different colors were used to distinguish biological process, cellular component, and molecular function.

As shown in Fig. 2B, many transport-related genes, such as VPS4A, RAB11FIP1, and ARF1 exhibited significant upregulation after the oral feeding with SPc, indicating the stronger cellular uptake with the aid of SPc. Meanwhile, the current study further focused on energy-related genes to illustrate the biotoxicity of SPc. Compared with the control, the exposure to SPc upregulated the expression levels of most energy-related genes, including TREH, FBP1, and ACC1. qRT-PCR was also performed to validate the expression levels of the above genes. The results were in accordance with the transcriptome data (Fig. 3). For example, VPS4A, RAB11FIP1, and TREH in nymphs treated with SPc were upregulated by 5.44-, 3.74-, and 17.79-fold compared with the control.

Fig. 3 Validation of the differentially expressed genes using quantitative real-time PCR. The relative mRNA levels of the target genes were normalized to the abundance of the Beta-actin gene. Asterisks indicate significant differences according to the independent t-test (*P < 0.05, **P < 0.01, and ***P < 0.001).

Loading capacity and automatic interaction of SPc with TTP

As shown in Table 2, the PLC of SPc toward TTP was calculated to be 32.2% using the freeze-drying method. The binding force of SPc with TTP was assessed using ITC (Fig. 4). According to the previous interpretation of ITC data, the negative ΔG (−8.94 kcal mol⁻¹) revealed that the self-assembly was automatic, and the high affinity constant K_a of 3.20 × 10⁵ M⁻¹ suggested that this interaction was strong. The negative values of ΔH (−2901 kcal mol⁻¹) and ΔS (−9.70 kcal mol⁻¹ deg⁻¹) suggested that the interaction between SPc and TTP might be driven primarily by hydrogen bonding and van der Waals force.

Table 2 Loading capacity of SPc toward TTP using the freeze-drying method

Weight of applied pesticide (mg)	Weight of applied SPc (mg)	Weight of pesticide-loaded complex (mg)	Weight of pesticide loaded in complex (mg)	Pesticide-loading content (%)
100	100	147.5	47.5	32.2

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Fig. 4 Schematic illustration of TTP/SPc complex formation (A) and ITC titration of SPc into TTP solution (B). The titration was performed by adding 38 μL of SPc solution (0.5 × 10⁻³ mol L⁻¹) to 200 μL of TTP solution (0.05 × 10⁻³ mol L⁻¹). The test temperature was 25 °C.

Reduced particle size and characterization of the TTP/SPc complex

As shown in Table 3 and Fig. 5A, the combination of TTP with SPc disturbed the self-aggregated structure of TTP in solution, decreasing its particle size from 2900 to 293 nm at a mass ratio of 1 : 1. Representative TEM images revealed that the self-aggregated TTP consisted of nearly spherical particles, while a significant portion of TTP/SPc complex exhibited a similar characterization with a much smaller particle size (Fig. 5B).

Table 3 Particle sizes of TTP and the TTP/SPc complex

Formulation	Mass ratio	Sample number	Size (nm)	Average size (nm)
Data were analyzed using the independent t test (P < 0.05).
TTP	—	1	2891.62	2876.53 ± 133.79
		2	2735.84
		3	3002.14
TTP/SPc complex	1 : 1	1	291.32	293.25 ± 2.16
		2	295.59
		3	292.85
T = 33.439, df = 4, P < 0.001

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Fig. 5 Particle-size distributions (A) and TEM images (B) of TTP/SPc complex at the mass ratio of 1 : 1. The tested concentration of TTP was 400 μg mL⁻¹.

Enhanced bioactivity of TTP/SPc complex against the common cutworm

The contact and stomach toxicity of TTP were improved significantly with the aid of SPc against the larvae of the common cutworm via the immersion and oral feeding methods, respectively (Fig. 6). For contact toxicity, the corrected mortalities were increased from 13% to 35% (1 mg L⁻¹), 25% to 48% (5 mg L⁻¹), and 32% to 66% (10 mg L⁻¹) at 2 d post treatment (Fig. 6A). Similarly, the corrected mortalities of the larvae fed with TTP/SPc complex were increased by 25% (1 mg mL⁻¹), 27% (5 mg L⁻¹), and 35% (10 mg L⁻¹) compared to TTP alone (Fig. 6B).

Fig. 6 Contact (A) and stomach toxicity (B) of SPc-loaded TTP against the 2nd instar larvae of the common cutworm. (A) Larvae were directly immersed in various formulations of TTP/SPc complex (mass ratio of 1 : 2.11; TTP concentration: 10, 5, and 1 mg L⁻¹), with ddH₂O used as the control. The number of dead larvae was recorded at 2 d after the treatment. Each treatment included 20 larvae, and was repeated 3 times. (B) Larvae were fed with artificial diets immersed in the above formulations.

High predation selectivity and non-toxicity of TTP/SPc complex toward the predatory stinkbug

As shown in Fig. 7A, the predatory stinkbug exhibited an obvious predation preference for alive larvae of the common cutworm with a predation rate of 83%. This pronounced preference for alive prey was beneficial for effective utilization of the predatory stinkbug. Furthermore, there was no significant difference in the survival rates of predatory stinkbugs treated with TTP/SPc complex and TTP alone (Fig. 7B).

Fig. 7 Cooperative pest control via the nano-pesticide and predatory stinkbug. (A) Predation selectivity of the predators. Five 2nd instar nymphs starved for 6 h were fed with 10 alive and 10 dead larvae. The predation preference of the nymphs was recorded at 24 h after the treatment. Each experiment was repeated 4 times. Asterisks indicate significant differences according to the independent t-test (***P < 0.001). (B) Survival rate of predators treated with the nano-pesticide. The newly-hatched nymphs and sterile cotton balls were immersed in various formulations of the nano-pesticide and the corresponding pesticide alone, respectively. The survival rate was recorded at 2 d after the treatment. Each treatment contained 20 nymphs and was repeated 4 times. “ns” indicates no significant differences according to the independent t-test. (C) Mortality of common cutworms in various treatments. All insects and artificial diets were immersed in corresponding solutions, respectively. Each treatment contained 40 larvae and was repeated 4 times.

Control effect of the TTP/SPc complex and predator co-application on the common cutworm

An effective cooperative pest control strategy was achieved via the co-application of the nano-pesticide and a predator. As shown in Fig. 7C, the mortality of larvae treated with the predator alone reached only 28%, indicating the relatively slow-acting property of the predator. Remarkably, the mortality was substantially increased to 88% following the co-application of the TTP/SPc complex and the predator. This co-application led to a 19% increase in mortality compared to the application of TTP/SPc complex alone.

Discussion

As a crucial natural enemy, the predatory stinkbug A. custos has been widely applied to control various agricultural and forestry pests.⁴⁴ It was important that the potential adverse impacts of SPc should be assessed before the extensive co-application of the predatory stinkbug and SPc-loaded pesticides. In the current study, the immersion of SPc showed no significant influences on the hatching rate of A. custos eggs. Consistent with prior research, SPc had negligible impacts on the hatching rate of ladybird eggs.²⁶ Insects fortify their eggshells by forming a chorion during the embryonic development, which might protect the embryo from SPc.⁴⁵ In the biotoxicity assay of SPc toward A. custos nymphs, SPc displayed no contact toxicity, but exhibited stomach toxicity with an LC₅₀ value of 14.75 mg mL⁻¹, indicating that the predatory stinkbug was more sensitive to the oral feeding of SPc. Similarly, the LC₅₀ values of SPc were 43 and 19 mg mL⁻¹ for contact and stomach toxicity against predatory ladybirds, respectively.²⁶ However, the concentration of SPc for field application is much lower than the LC₅₀ values for predators, implying that the SPc is nontoxic to predators at the working concentration.

To further elucidate the lethal mechanism of SPc at extremely high concentration, we conducted RNA-seq analysis and identified some DEGs following SPc treatment. In particular, many transport-related genes were upregulated after the oral feeding with SPc. Among these genes, the Vacuolar protein sorting-associated protein 4A (VPS4A) gene is a critical participant in transporting proteins out of a perivacuolar endosomal compartment, which is involved in the late steps of the endosomal multivesicular body pathway.^46,47 Also, the Rab11 family-interacting protein 1 (RAB11FIP1) gene is notable and is responsible for membrane trafficking along the phagocytic pathway and in phagocytosis.⁴⁹ The upregulation of these genes revealed the stronger cellular uptake, which is consistent with our previous findings that SPc can activate clathrin-mediated endocytosis.^48,49

The exposure of SPc can alter the expression levels of key genes related with membrane protein, drug metabolism, apoptosis, etc.^26,50 Compared with the control, oral feeding with SPc upregulated most energy-related genes in the predatory stinkbug. For example, trehalase (TREH) gene encoding intestinal trehalase is involved in the hydrolysis of ingested trehalose.^51,52 The folate-binding protein 1 (FBP1) gene plays a vital role in regulating glucose sensing and the insulin secretion of pancreatic beta-cells.⁵³ The acetyl-CoA carboxylase 1 (ACC1) gene catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, which is involved in fatty acid synthesis.^54,55 Based on the above RNA-seq results, we deduced that the SPc at extremely high concentration acted as a stressor, which augmented the processes of phagocytosis, exocytosis, and energy synthesis in A. custos. The higher metabolic activity could be expected to cause substantial energy expenditure, ultimately culminating in the death of the predatory stinkbug. Similar with a previous study, the oral feeding of SPc resulted in a significant downregulation of many membrane protein genes, while upregulating most genes encoding small ribosomal subunit proteins in predatory ladybirds.²⁶ In addition, SPc exhibited a predilection for accumulation within intestinal cells to concurrently induce systemic responses in insects.⁵⁵

As an efficient insecticide toward lepidopteran pests, TTP acts on the ryanodine receptor to result in the release of a large amount of intracellular calcium ions, leading to insect death due to muscle contraction and paralysis.^30,56 Meanwhile, TTP displays good selectivity to insect pests and non-target organisms, which indicates it is relatively eco-friendly under a reasonable dosage.⁵⁷ The current study aimed to construct a nontoxic TTP nano-pesticide toward the predatory stinkbug with the help of SPc, and to co-apply the TTP nano-pesticide and predatory stinkbug for perfect cooperative pest management. SPc can self-assemble with various insecticides, such as imidaclothiz, methoxyfenozide, and thiamethoxam, to enhance their bioactivity.^23,24,37 In the current study, it was found that SPc could assemble with TTP into TTP/SPc complex via hydrogen bonding and van der Waals force, and the PLC was calculated to be 32.2%, which was relatively higher than those toward thiamethoxam (20.63%), cyantraniliprole (24.79%), and monosultap (19.3%).^23,58–60 The SPc can combine with an insecticide via various interactions, such as electrostatic interaction, hydrophobic interaction, and hydrogen bonding.^23,24,61 The dominant interaction forces are usually dependent on the chemical structures, particularly the functional groups of the SPc and pesticides. The diversity of functional groups in SPc is conducive to expanding the application field of SPc.

The complexation with SPc broke the self-aggregated TTP and reduced its particle size down to 293 nm. Consistent with previous findings, SPc can encapsulate various pesticide molecules within its cavities, achieving pesticide nanometerization. With the help of SPc, the particle sizes of imidaclothiz, osthole, and thiocyclam were reduced to 84.28, 17.66, and 52.74 nm, respectively.^24,60,62 The pesticide nanometerization offers several advantages, including an amplified contact area, stronger membrane penetration, enhanced systemic transmission, and improved bioactivity. In the current study, the TTP/SPc complex displayed stronger toxicity against the common cutworm through immersion and oral feeding methods. Similar to a previous study, SPc-loaded avermectin showed stronger stomach and contact toxicity against green peach aphids, with mortality increases of 32.7% and 27.8%, respectively.²⁵ Furthermore, the LC₅₀ value of osthole was decreased from 0.05 to 0.03 g L⁻¹ against aphids and from 0.33 to 0.27 g L⁻¹ against mites after the complexation with SPc.⁶² The possible explanation for the enhanced toxicity may be the pesticide nanometerization amplifying the contact area of the pesticide. Moreover, the nanocarrier could promote the cellular processes of endocytosis and exocytosis, facilitating the active transportation of pesticide into the cells.

Although the bioactivity of TTP was improved against target pests with the aid of SPc, the current study confirmed the application safety of SPc-loaded TTP toward predatory stinkbugs. The predators displayed an obvious predation preference for alive common cutworm, which not only reduced the exposure risk of the predator to the nano-pesticide, but also was advantageous for better control effects on the pests. The predatory stinkbug rely on its antennae to detect various chemical signals, and living prey typically generate movement and vibrations, which can attract the attention of predatory stinkbugs seeking suitable prey.⁶³ More importantly, the TTP/SPc complex showed no significant stomach or contact toxicity against the predatory stinkbug. The insensitivity of the predatory stinkbug to TTP may be due to the binding site difference between the pest and predator, and TTP has a unique chemical structure and displays excellent activity against lepidopterans.^64,65 Consequently, TTP/SPc complex could be co-applied with the predatory stinkbug for perfect cooperative pest management. Compared to the application of TTP or the predatory stinkbug alone, the advantage of co-application was derived from the higher biological activity of the TTP nano-pesticides against pests. A low dosage of TTP could achieve an excellent control effect, which exhibited no adverse effects on predators. Similar to a previous research, the co-application of nano-pesticides and predatory ladybirds could achieve the sustainable management of green peach aphids.²⁶

Conclusions

In summary, we introduced a nanocarrier SPc to construct a TTP nano-pesticide, which was successfully co-applied with the predatory stinkbug A. custos for cooperative pest control. We first demonstrated the biosafety of SPc at the working concentration toward the predatory stinkbug. However, the oral feeding of SPc at extremely high concentrations induced multiple stress responses in the predator, which augmented the processes of phagocytosis, exocytosis, and energy synthesis that ultimately led to its death. Next, the TTP was incubated with SPc to prepare the TTP/SPc complex, and its self-assembly was driven primarily by hydrogen bonding and van der Waals force, which reduced the particle size of TTP down to the nanoscale. Impressively, the contact and stomach toxicity of TTP/SPc complex were remarkably higher than TTP alone against the common cutworm due to the amplified contact area of the nano-pesticide. Interestingly, the predatory stinkbug exhibited a strong predation preference for alive common cutworm and was insensitive to the nano-pesticide, which reduced the exposure risk of the predators to the nano-pesticide. Thus, the co-application of the nano-pesticide and predatory stinkbug could achieve efficient pest control. Overall, our study developed a nano-pesticide that was nontoxic toward the predatory stinkbug, which provided a perfect example for cooperative pest control and pesticide reduction via co-application of the nano-pesticide and predator.

Data availability statement

Data will be made available on request.

Author contributions

Shangyuan Wu: investigation, methodology, data curation. Qinhong Jiang: investigation, methodology, data curation. Chunyang Huang: investigation, methodology, data curation. Hailin Yang: methodology. Changhua Zhang: methodology. Meizhen Yin: methodology. Jie Shen: methodology. Shuo Yan: conceptualization, supervision, funding acquisition, writing – review & editing. Hu Li: conceptualization, supervision, funding acquisition.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the China National Tobacco Corporation of Science and Technology Major Projects (No. 110202201018[LS-02]), Key Project of Science and Technology Plan of Yunnan Company of China National Tobacco Corporation (2022530000241021), National Natural Science Foundation of China (32372631) and the 2115 Talent Development Program of China Agricultural University.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4en00060a

‡ These authors have contributed equally to this work.

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