Lethal Effect of Pulsed Electric Fields on Tribolium castaneum: Optimization and Mechanistic Insight into Electro-Neurotoxicity

Abstract

To address the issues of resistance and food safety stemming from the overuse of chemical fumigants in stored-grain pest control, this study aimed to systematically optimize the insecticidal process of pulsed electric field (PEF) treatment on Tribolium castaneum (T. castaneum) and to investigate its electro-neurotoxicity mechanism. Single-factor experiments were used to determine parameter ranges, and response surface methodology (RSM) was employed to analyze the effects of electric field strength, pulse frequency, and treatment time. The finite element method (FEM) was used to simulate the physical field distribution, and acetylcholinesterase (AChE) activity was measured to explore neurotoxicity. The results indicated that electric field strength, pulse frequency, and treatment time all had highly significant effects (p < 0.0001), with electric field strength being the primary factor. The optimal process parameters were determined to be: electric field strength of 26 kV/cm, pulse frequency of 20 kHz, and treatment time of 140 s. Under these conditions, the predicted and actual mortality rates were both 100%, and this efficacy was validated in rice samples. Simulation confirmed that PEF achieves physical targeting through a “tip effect” on the insect’s nerve endings; mechanism tests demonstrated that PEF treatment significantly inhibited AChE activity (p < 0.01). This study confirms the “electro-neurotoxicity” mechanism of PEF, providing theoretical support for this green physical control technology.

1. Introduction

Food security stands as the cornerstone of national stability and social development. However, in the global production, transportation, and storage of grain, losses caused by stored-product pests reach an average annual rate of 5–10%, and this figure is even higher in developing countries [1,2]. Beyond quantitative weight loss, infestation leads to significant qualitative degradation—such as nutritional loss and contamination via secretions and insect fragments—resulting in substantial economic deficits estimated at billions of dollars annually [3]. Tribolium castaneum (T. castaneum), a cosmopolitan and highly destructive stored-product pest, poses a serious threat to various stored commodities such as wheat, rice, corn, and their products, owing to its high reproductive capacity, short life cycle, and wide adaptability [4]. Severe infestation not only facilitates mold growth but also leads to the accumulation of carcinogenic quinone secretions (benzoquinones), posing potential health risks to consumers [5,6].

Currently, the control of stored-product pests relies heavily on chemical fumigation, particularly phosphine [7]. Although effective and cost-efficient, the negative consequences of its long-term, large-scale use are increasingly prominent. Firstly, pest resistance is intensifying; high-level resistance to phosphine has been reported in T. castaneum populations globally, significantly reducing control efficacy [8]. This reduced efficacy exacerbates economic losses and compromises grain marketability, making pest management increasingly difficult and costly [9]. Secondly, chemical residues and toxicity pose multi-dimensional risks. Beyond the direct threat to food safety and the ecological environment, the application of toxic fumigants presents severe occupational health risks to operators [10]. Furthermore, regulatory bodies worldwide are enforcing increasingly stringent Maximum Residue Limits (MRLs) for chemical pesticides [11], restricting their usage. Therefore, developing novel, green, and environmentally friendly physical control technologies is an urgent priority for ensuring food security and sustainable agricultural development.

Various physical disinfestation technologies, such as radio frequency (RF), microwave (MW), and cold plasma (CP), have been investigated as alternatives to chemical fumigation. However, these methods exhibit certain limitations; RF and MW treatments are often associated with thermal effects and non-uniform heating, which may compromise grain quality, while CP treatment is typically restricted by its limited penetration depth, affecting its efficacy in bulk grain [12,13]. In contrast, among emerging physical processing technologies, pulsed electric field (PEF) technology has attracted widespread attention from both academia and industry due to its distinct non-thermal effects, high efficiency, and low energy consumption [14,15]. This technology involves applying high-voltage microsecond or nanosecond pulses between two electrodes, generating a high-intensity electric field within the treated material. Its core mechanism is widely believed to be the electroporation effect, where the strong electric field transiently increases the permeability of cell membranes, forming reversible or irreversible pores. This leads to the leakage of intracellular contents, metabolic disruption, and ultimately, cell death [16,17].

Based on this principle, PEF technology has been successfully applied in the food industry, for instance, in the non-thermal sterilization of liquid foods like juice and milk to maximize nutrient and flavor retention [18,19,20], and as an auxiliary method for cell disruption to enhance the extraction of bioactive compounds [21]. These established applications demonstrate that PEF, as a technology with potent biological effects, possesses immense potential to disrupt cellular structures and impair physiological functions.

Building on these cellular insights, researchers have extended PEF investigations to multicellular organisms, confirming it induces physiological stress in pests [22,23,24]. For stored-product insects, El Hajj et al. [25] highlighted a critical challenge: “latent vitality,” where insects recover from temporary paralysis. This phenomenon indicates that arbitrary parameter settings often fail to ensure irreversible mortality. Therefore, systematic process optimization is essential not only to overcome this resistance but also to determine the most cost-effective combination that maximizes lethality while minimizing energy inputs. Moreover, this “knockdown-recovery” pattern implies neurological impact rather than mere physical damage. Yet, PEF application on T. castaneum remains underexplored, and the potential “electro-neurotoxicity” pathway mediated by the inhibition of acetylcholinesterase (AChE) has not been verified as the driver of this lethality.

To deeply understand this mechanism, it is necessary to explore how the electric field induces physiological dysfunction. In classical toxicology, the nervous system is the primary target of most chemical insecticides (e.g., organophosphates) [26]. This raises a critical scientific question: Does PEF treatment inhibit key enzymes in the insect nervous system, such as AChE, similarly to chemical agents? Investigating this potential “electro-neurotoxicity” pathway is crucial for elucidating the unique mechanism of action of PEF.

Against this background, this study focuses on the typical stored-product pest T. castaneum, with the aim of systematically integrating process optimization with the investigation of the electro-neurotoxicity mechanism. To address the gap in process optimization, this study first explores the influence of various parameters through single-factor experiments. Subsequently, response surface methodology (RSM) is employed to analyze the interactions between factors to identify the optimal insecticidal parameters. To address the gap in mechanistic understanding, the finite element method (FEM) is used to simulate the electric field distribution and reveal the “tip effect” and “focusing effect” from a physical perspective. Additionally, AChE activity is measured to verify the lethal pathway from a biochemical (neurotoxic) perspective. Through a comprehensive analysis of both efficacy (process optimization) and mechanism (electro-neurotoxicity), this study provides a solid theoretical foundation for the application of PEF technology as a green, physical control method for stored-product pests.

2. Materials and Methods

2.1. Experimental Materials and Instruments

2.1.1. Experimental Insects and Materials

The Tribolium castaneum (T. castaneum) used in this study were originally obtained from Wotian Breeding Family Farm (Si County, Anhui, China) and were subsequently reared in our laboratory for multiple generations to establish a stable colony. The rearing conditions were maintained at 28 ± 2 °C and 68 ± 2% relative humidity in constant darkness. The diet consisted of a mixture of whole wheat flour and yeast powder (95:5, w/w) [27]. To ensure consistency, active adults of uniform size (1–2 weeks post-eclosion) were selected for each experiment. The inclusion criteria required insects to have intact appendages and exhibit normal motility, while individuals showing physical defects or sluggish movement were excluded. They were randomly assigned to treatment groups to minimize systematic errors. The rice used for validation was purchased from Dizhijin Rice Co., Ltd. (Wuchang, China).

2.1.2. Main Instruments and Reagents

The experimental setup (Figure 1) included a microsecond pulse power supply (CTP-2000K, Nanjing Suman Plasma Technology Co., Ltd., Nanjing, China), a constant temperature and humidity chamber (BPHS-060B, Shanghai Boxun Medical Biological Instrument Corp., Shanghai, China), a digital microscope (AD3800, Shenzhen Andonstar Tech Co., Ltd., Shenzhen, China), and an infrared thermal imager (UTi260A, Uni-Trend Technology (China) Co., Ltd., Dongguan, China). Biochemical analyses utilized a UV-visible spectrophotometer (UV-1800, AOE Instruments Co., Ltd., Shanghai, China), a high-speed refrigerated centrifuge (TGL-16M, Hunan Xiangyi Laboratory Instrument Development Co., Ltd., Changsha, China), and a vortex mixer (XH-D, Woxin Instrument Co., Ltd., Wuxi, China). The acetylcholinesterase (AChE) assay kit (BC2020) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

2.2. PEF Experimental Platform

The experimental platform established in this study is illustrated in Figure 2. The core equipment was a microsecond pulse power supply that provided an output voltage amplitude of 0–25 kV, a frequency of 5–20 kHz, and a pulse width of 1 μs, and was characterized by a steep rising edge. The processing chamber comprised two rectangular parallel plate electrodes made of 304 stainless steel (30 cm × 20 cm × 0.5 cm) with an adjustable gap. In this study, the distance between the electrodes was fixed at 0.5 cm. Experimental samples were placed in a 50 mm diameter polystyrene Petri dish, which was positioned at the geometric center of the parallel plates to ensure full exposure of T. castaneum to the uniform electric field. To ensure environmental stability and reproducibility, the electrode assembly was housed in a constant temperature and humidity chamber maintained at 28 ± 2 °C and 68 ± 2% relative humidity, while the power supply was operated externally.

2.3. Experimental Methods

Rationale for Frequency Selection: The selection of the kilohertz frequency range (e.g., 20 kHz) is theoretically grounded in the distinct dielectric properties of the target pest and the grain medium. Dry rice grains act as low-loss dielectrics (insulators), whereas T. castaneum adults, possessing conductive body fluids, behave as lossy dielectrics (conductors). According to the Maxwell-Wagner polarization theory, in a heterogeneous system exposed to low-to-medium frequency electric fields, charge accumulation occurs at the interfaces between materials with different conductivities [28]. At 20 kHz, this interfacial polarization is maximized, allowing the electric field to effectively bypass the insulating grain matrix and concentrate selectively within the insect body. This theoretical assumption ensures deep penetration and targeted energy delivery, minimizing energy waste on the grain.

2.3.1. Preliminary Experiments and Factor Level Determination

Preliminary experiments were conducted to explore the influence of key PEF parameters on the mortality of T. castaneum and to determine the appropriate factor ranges for the subsequent response surface optimization. The corrected mortality rate was used as the response variable. Throughout the preliminary tests, the natural mortality in the control group was monitored and remained consistently low (ranging from 0% to 5%), ensuring the validity of the correction. To systematically evaluate the parameter effects, the experiments were carried out according to the following protocols:

Effect of electric field strength and treatment time: With the pulse frequency fixed at 20 kHz, a full factorial experiment was conducted combining electric field strengths of 16, 20, 24, and 28 kV/cm with treatment times of 30, 60, 180, and 300 s.

Effect of pulse frequency: With the treatment time fixed at 300 s, the effect of pulse frequency (5, 10, 15, and 20 kHz) was evaluated across different electric field strengths (16, 20, 24, and 28 kV/cm).

2.3.2. Response Surface Methodology (RSM) Design

Based on the preliminary results, a three-factor, three-level Box–Behnken Design (BBD) [29] was employed to optimize the insecticidal process. The three independent variables chosen were electric field strength (A), pulse frequency (B), and treatment time (C). The experimental ranges and levels were explicitly set as follows: A ranging from 20 to 28 kV/cm (20, 24, 28 kV/cm); B ranging from 10 to 20 kHz (10, 15, 20 kHz); and C ranging from 60 to 300 s (60, 180, 300 s). The design consisted of 17 randomized experimental runs, comprising 12 factorial points and 5 center points to estimate pure error (

Table 1. Experimental design and results of the BBD for T. castaneum mortality.

No.	Electric Field Strength/(kV/cm)	Frequency/(kHz)	Processing Time/(s)	Mortality Rate/(%)
1	20	10	180	84
2	28	10	180	100
3	20	20	180	96
4	28	20	180	100
5	20	15	60	80
6	28	15	60	100
7	20	15	300	96
8	28	15	300	100
9	24	10	60	86
10	24	20	60	96
11	24	10	300	96
12	24	20	300	100
13	24	15	180	92
14	24	15	180	96
15	24	15	180	96
16	24	15	180	96
17	24	15	180	96

). The corrected mortality rate was used as the response value (R).

2.3.3. Mortality Assessment and Calculation

For each parameter combination, 100 T. castaneum adults were selected and divided into 4 groups: 3 replicates (25 insects each) for the treatment group, and 1 group (25 insects) as the control. The control group was maintained in the same environment but without electric field exposure. After treatment, all samples were returned to the rearing environment and observed every 6 h until 24 h post-treatment. An insect was considered dead if its appendages showed no reaction when the junction of the elytra was lightly touched with a soft brush. This criterion was strictly applied to exclude thanatosis (death-feigning). The mortality rate was calculated using Equation (1):

$$M={\displaystyle \frac{N_d}{N_{total}}}\times 100\%$$

(1)

where M is the mortality rate (%), N_d is the number of dead individuals in the treatment group, and N_total is the total number of individuals in the treatment group.

To account for natural death, the mortality was corrected using Abbott’s formula [30] (Equation (2)).

$$CM={\displaystyle \frac{M_t-M_c}{100-M_c}}\times 100\%$$

(2)

where CM is the corrected mortality rate (%), M_t is the observed mortality rate in the treatment group (%), and M_c is the mortality rate in the control group (%).

2.3.4. Electric Field Simulation

To investigate the electric field distribution within the processing chamber, a finite element simulation was performed using COMSOL Multiphysics 6.3. A 3D geometric model consistent with the experimental platform was established. Crucially, to accurately reflect the granular nature of the sample, the processing domain was modeled as a heterogeneous mixture rather than a dense, homogeneous layer. It explicitly included randomly distributed rice grains, air voids (porosity), and the target T. castaneum located within the grain interstices. The electrodes were defined as 304 stainless steel, while air and T. castaneum were assigned appropriate relative permittivities and electrical conductivities [28]. The “Electric Currents” interface in the AC/DC Module was selected. A high voltage was applied to the upper electrode, while the lower electrode was grounded. A free triangular mesh was used for discretization, with refinement focused on the insect-containing regions. Finally, a stationary study was conducted to obtain the electric field magnitude plot.

2.3.5. AChE Activity Assay

Treated insects from specific experimental groups and untreated insects from the control group were collected for enzyme activity assays 24 h post-treatment. To evaluate the physiological response across the experimental design boundaries, samples were specifically selected from treatment groups representing the minimum and maximum levels of the key parameters: electric field strength (16 and 28 kV/cm), pulse frequency (5 and 20 kHz), and treatment time (30 and 300 s).

Twenty insects were randomly selected from each group, accurately weighed (W), and placed in pre-cooled 1.5 mL microcentrifuge tubes. Subsequently, 100 μL of extraction buffer was added, and the samples were thoroughly ground for 2 min in an ice bath using the grinding rod provided with the AChE assay kit. Then, an additional 900 μL of extraction buffer was added, and the mixture was homogenized by vortexing at 2500 rpm for 1 min using a vortex mixer. The homogenate was centrifuged at 8000× g for 10 min at 4 °C, and the supernatant was collected as the crude enzyme solution. The assay was performed strictly according to the instructions of the AChE assay kit. The absorbance was measured at 412 nm using a spectrophotometer. The AChE activity was calculated using Equation (3) [31,32]:

$$\mathrm{AChE} \mathrm{Activity} ={\displaystyle \frac{2250\times \Delta A}{W}}$$

(3)

where ∆A is the change in absorbance (difference between the measurement and the control), W is the weight of the sample (g), and 2250 is the calculation coefficient derived from the reaction system parameters (including total volume, reaction time, and extinction coefficient) as standardized by the assay kit.

2.4. Data Statistical Analysis

All experiments were performed in triplicate, and results are expressed as mean ± standard deviation (SD). For the preliminary experiments and the determination of key process parameters, data were processed using descriptive statistics (Origin 2022) to evaluate response trends. In the case of AChE activity assays, differences between treatment groups and the control were analyzed using one-way Analysis of Variance (ANOVA) followed by Dunnett’s test (p < 0.05), with prior verification of normality (Shapiro–Wilk test) and homogeneity of variance (Levene’s test). For the RSM study, Design-Expert 13.0 was utilized for experimental design and regression analysis, where model adequacy was rigorously validated through diagnostic plots.

3. Results

3.1. Experimental Validation of PEF Insecticidal Process

To verify the feasibility of the PEF insecticidal process, preliminary experiments were conducted to observe the general trends of mortality. The results at a fixed frequency are presented in Figure 3a. With the frequency fixed at 20 kHz, extending the treatment time from 30 s to 300 s resulted in a gradual increase in the 24 h mortality rate across electric field strengths of 16–24 kV/cm. Notably, at 28 kV/cm, 100% mortality was achieved after only 30 s of exposure. The results at a fixed treatment time (300 s) are shown in Figure 3b. Within the 16–20 kV/cm range, mortality increased with frequency (5–20 kHz). At 24 kV/cm, 100% mortality was attained even at the lowest frequency of 5 kHz. These preliminary findings confirm that electric field strength, frequency, and treatment time are all critical factors affecting the efficacy of PEF treatment.

3.1.1. Influence of Key Process Parameters on T. castaneum

Based on the preliminary validation, single-factor experiments were further analyzed to determine the optimal parameter ranges for response surface methodology (RSM) optimization (Figure 4).

Electric Field Strength: Under the conditions of 180 s treatment time and 20 kHz frequency (Figure 4a), the mortality rate was only 26% at 16 kV/cm. However, in the 20–28 kV/cm range, mortality rates consistently exceeded 95%. Therefore, 20–28 kV/cm was selected as the optimization range.

Pulse Frequency: With the treatment time fixed at 300 s and electric field strength at 20 kV/cm (Figure 4b), mortality rates exceeded 80% at frequencies above 10 kHz. Consequently, the frequency range of 10–20 kHz was chosen.

Treatment Time: Under conditions of 20 kHz and 20 kV/cm (Figure 4c), the mortality rate remained above 70% when the treatment time exceeded 60 s. Thus, the range of 60–300 s was selected for the optimal process search.

3.1.2. Establishment and Evaluation of the Mortality Response Surface Model

To explore the interactions between the process parameters and to determine the most cost-effective combination for efficient extermination of T. castaneum, this study employed the Box–Behnken Design (BBD) method for a three-factor, three-level response surface optimization experiment based on the ranges determined above. The experimental design and results are shown in Table 1.

Multiple regression fitting was performed to obtain a polynomial regression model including linear and interaction terms for the mortality rate (R) as a function of electric field strength (A), pulse frequency (B), and treatment time (C).

$$R=5.5\times A+3.25\times B+3.75\times C-3\times AB-4\times AC-1.5\times BC+94.71$$

(4)

where A is the electric field strength, B is the frequency, C is the treatment time, and R is the mortality rate.

Prior to the ANOVA, the model assumptions were verified using diagnostic plots. The normal probability plot of residuals followed a straight line, and the plot of residuals versus predicted values showed a random distribution, confirming that the assumptions of normality and homogeneity of variance were met. To evaluate the significance and goodness-of-fit of the established model, an ANOVA was performed on the regression model, with results shown in

Table 2. Results of ANOVA.

Source	Sum of Squares	df	Mean Square	F-Value	p-Value
model	548.00	6	91.33	46.77	<0.0001
A	242.00	1	242.00	123.92	<0.0001
B	84.50	1	84.50	43.27	<0.0001
C	112.50	1	112.50	57.61	<0.0001
AB	36.00	1	36.00	18.43	0.0016
AC	64.00	1	64.00	32.77	0.0002
BC	9.00	1	9.00	4.61	0.0574
Residuals	19.53	10	1.95	-
Lack of Fit	6.73	6	1.12	0.3505	0.8786
Pure Error	12.80	4	3.20	-	-
Total	567.53	16	-	-	-

.

Table 2 summarizes the ANOVA results. The model F-value of 46.77 and p-value of < 0.0001 indicate that the model is highly significant. The “Lack of Fit” p-value was 0.8786 (>0.05), suggesting the model fits the experimental data well. The linear terms A, B, and C were all significant (p < 0.0001). Based on the F-values (F_A = 123.92, F_C = 57.61, F_B = 43.27), the order of influence was Electric Field Strength A > C > B.Regarding interactions, AB (p = 0.0016) and AC (p = 0.0002) were significant, whereas BC (p = 0.0574) was not.

The model fit statistics (

Table 3. Model fitting statistics table.

Statistical Indicators	Numeric Value	Fitting Indicators	Numeric Value
Std. Dev.	1.40	R2	0.9656
Adeq Precision	94.71	Adjusted R2	0.9449
C.V.%	1.48	Predicted R2	0.9222

) further validate reliability. The coefficient of determination (R² = 0.9656) and adjusted R² (0.9449) are close to 1, and the predicted R² (0.9222) confirms good predictive capability. The coefficient of variation (C.V.%) was 1.48%, indicating high precision. The Adeq Precision (94.71) indicates an excellent signal-to-noise ratio.

3.1.3. Analysis of Process Parameter Interactions

To visualize the interactions, 3D response surface and contour plots were generated. Figure 5 illustrates the interaction between A and C. The steep slope of the response surface and the elliptical contour lines indicate a significant synergistic effect. While increasing one factor at a low level of the other had a limited effect, simultaneous high levels of both resulted in mortality rapidly approaching 100%.

Similarly, Figure 6 shows the interaction between A and B. The significant interaction suggests that at higher electric field strengths, increasing the pulse frequency effectively enhances mortality. In contrast, the non-significant BC interaction implies that the effects of frequency and time are largely independent within the tested range.

3.1.4. Selection and Validation of Optimal Process Parameters

Numerical optimization was performed to achieve maximum mortality with minimum energy input (minimized field strength and time). Specifically, the optimization criteria prioritized selecting the minimum electric field strength necessary to achieve 100% mortality. This strategy was employed to ensure sufficient lethality while maintaining a safety margin to keep the process within a safer range below the theoretical air breakdown strength (~30 kV/cm). Based on these constraints, the optimal parameters were predicted to be: 26 kV/cm, 20 kHz, and 140 s, with a predicted mortality of 99.59% (Figure 7).

Validation experiments (n = 3) conducted under these conditions yielded an actual mortality of 100%, confirming the model’s accuracy. Furthermore, to simulate real-world conditions, validation was performed with T. castaneum mixed in rice (25 insects/25 g). The mortality in the rice mixture also reached 100%, demonstrating that PEF treatment can effectively penetrate grain interstices to control hidden pests. Figure 8 shows the insect condition before and after PEF treatment.

3.2. Electric Field Distribution Simulation and Its Correlation with Insecticidal Effect

To elucidate the physical mechanism of the PEF lethal effect on T. castaneum and to provide a theoretical basis for the experimental results observed in Section 3.1 (specifically, the efficacy of “in-rice validation” and the dominance of electric field strength), a finite element simulation was conducted under the optimal conditions (26 kV/cm). The simulation results (Figure 9) reveal two key characteristics of the electric field distribution:

First, the electric field exhibits “macroscopic penetration” (Figure 9a,b). As shown in the overall chamber distribution (Figure 9a) and the surface field of the rice-insect mixture (Figure 9b), the applied electric field can effectively penetrate the voids between rice grains. Even within the grain bulk, the field strength in the areas occupied by T. castaneum maintains a level sufficient to be lethal. This finding provides direct physical evidence supporting the 100% mortality rate observed in the rice validation experiments (Section 3.1.4), confirming the potential of PEF technology for practical application in stored-grain environments.

Second, the electric field demonstrates “microscopic targeting” (the “tip effect”) (Figure 9c). Due to the significant difference in electrical properties (permittivity) between T. castaneum and the surrounding air/rice, the electric field lines are significantly distorted and concentrated on the insect’s body. Critically, as shown in the zoomed-in view (Figure 9c), this concentration is dramatically amplified at sharp anatomical structures, such as the head, tail, antennae, and legs. This “tip effect” causes the local field strength in these nerve-ending-rich areas to far exceed the applied average field strength (26 kV/cm), creating “vulnerable sites” that PEF preferentially attacks.

These simulation results jointly elucidate why electric field strength is the primary factor governing lethality. The lethal mechanism generally follows the theory of cell electroporation, which posits the existence of a “lethal threshold” for the electric field strength required to induce membrane permeabilization [33,34,35]. It is important to note that while the minimum threshold for reversible or initial irreversible electroporation in a single cell suspension is typically around 0.5–2 kV/cm, a significantly higher external electric field is required to achieve complete lethality in a complex biological organism like T. castaneum. This is because the electric field distribution in tissues is non-uniform and undergoes attenuation. Therefore, the applied 26 kV/cm field strength is necessary to ensure that, after passing through the rice medium and being amplified by the tip effect, the local field strength at target cells (especially nerve endings) exceeds the irreversible electroporation threshold, thereby inducing sufficient cellular damage and ultimately leading to mortality. This explains the threshold-dependence of the mortality rate: below the threshold, efficacy is negligible; once exceeded, mortality rapidly saturates. Therefore, a sufficiently applied electric field strength is the fundamental physical prerequisite for triggering the subsequent biological cascade reactions (e.g., acetylcholinesterase (AChE) inactivation) discussed in the following section.

3.3. Evaluation of Thermal Effects

While PEF is fundamentally considered a non-thermal processing technology, the application of PEF can inevitably generate Joule heat within the treated samples. This is particularly relevant under the conditions employed in this study, which include relatively high frequencies (e.g., up to 20 kHz) and longer treatment durations (e.g., up to 300 s). Therefore, to definitively exclude the contribution of thermal effects to the observed insecticidal action, a dedicated experiment was performed.

As detailed in Section 2.3.1, this dedicated experiment was performed under the most stringent PEF conditions (electric field strength of 28 kV/cm, frequency of 20 kHz, 600 s total duration on a 25 g rice sample) to rigorously assess thermal effects. The results showed that, even after 600 s of continuous PEF application, the maximum temperature increase on the sample surface, relative to the initial ambient temperature (28 ± 1 °C), was less than 1.5 °C. This extremely minimal temperature rise strongly indicates that the observed insecticidal effects primarily originate from non-thermal mechanisms such as electroporation, with any thermal contribution being negligible.

3.4. Neurotoxic Mechanism of PEF for Pest Control

To reveal the lethal mechanism of PEF at the biochemical level, the activity of AChE, a key neurotransmitter-hydrolyzing enzyme, was measured in T. castaneum under different treatment conditions. The results are presented in Figure 10. The basal AChE activity in the control group was 21,066.41 ± 159.54 Units per gram (U/g). Compared to the control, AChE activity in all PEF-treated groups was significantly inhibited (p < 0.01), showing a clear dose-dependent relationship with the treatment parameters. Notably, this biochemical inhibition was consistent with the observed physical symptoms: while control insects remained active, treated individuals—especially in the high-intensity groups—exhibited distinct neurotoxic behaviors such as sluggishness, tremors, and immobility prior to sampling.

Effect of Electric Field Strength (Figure 10a; fixed at 20 kHz, 300 s): Increasing the electric field strength from 16 kV/cm to 28 kV/cm caused a sharp decline in AChE activity from 11,253.93 ± 511.12 U/g to 6039.37 ± 344.20 U/g, indicating a substantial increase in inhibition.

Effect of Pulse Frequency (Figure 10b; fixed at 28 kV/cm, 300 s): Increasing the pulse frequency from 5 kHz to 20 kHz resulted in a significant decrease in AChE activity from 7464.94 ± 129.08 U/g to 6039.37 ± 344.20 U/g.

Effect of Treatment Time (Figure 10c; fixed at 28 kV/cm, 20 kHz): Extending the treatment time from 30 s to 300 s led to a further reduction in AChE activity from 7265.63 ± 241.23 U/g to 6039.37 ± 344.20 U/g.

These results indicate that PEF treatment can significantly inhibit AChE activity in T. castaneum, and this inhibitory effect is intensified with increases in electric field strength, frequency, and treatment time. This strongly demonstrates that the lethal effect of PEF is mediated, at least in part, by disrupting the nervous system function via AChE inactivation.

4. Discussion

4.1. Electro-Neurotoxicity Mechanism: From Macroscopic Field to Molecular Targets

By integrating the results of process optimization, electric field simulation, and enzyme activity assays, a comprehensive lethal pathway linking the macroscopic physical field to microscopic molecular targets is proposed (Figure 11). This pathway is logically deduced as follows: It is crucial to note that this proposed mechanism primarily stems from non-thermal effects, as dedicated experiments (Section 3.3) have confirmed that the temperature increase during PEF treatment is negligible, thus excluding significant thermal contributions to the insecticidal action.

Before detailing the pathway, the rationale for the applied parameters must be clarified, particularly regarding the risk of air breakdown. It is acknowledged that the optimal electric field strength determined in this study (26–28 kV/cm) approaches the theoretical dielectric breakdown strength of air (~30 kV/cm). However, the selection of this high-intensity range was a necessary prerequisite for efficacy. As revealed by the simulation (Figure 9), the lethal mechanism relies on the “focusing effect” to amplify the electric field at the insect’s anatomical extremities. Only when the macroscopic applied field reaches this sufficient level can the localized field strength at the vulnerable sites exceed the critical threshold for irreversible electroporation.

1.

Initiation via Physical Stress. This study determined the optimal insecticidal parameters via response surface methodology (RSM) (26 kV/cm, 20 kHz, 140 s). Simulation results (Section 3.2) confirmed that under these conditions, due to the disparity in dielectric constants between the Tribolium castaneum (T. castaneum) body and the air, the electric field exhibited a significant “focusing effect.” Specifically, the local field strength was intensified at the head and tail regions—where nerve endings are concentrated—far exceeding the applied average field strength.

2.

Cellular Trigger via Electroporation. The lethal effect of PEF initiates with cellular electroporation. The localized high field strength described above is believed to exceed the electroporation threshold of nerve cell membranes in these regions [33]. This creates transient or irreversible micropores in the membrane, serving as the physical “trigger” for all subsequent biochemical damage.

3.

Biochemical Cascades and acetylcholinesterase (AChE) Inactivation. Electroporation immediately disrupts cellular homeostasis, triggering catastrophic biochemical cascades that directly or indirectly lead to AChE inactivation. First, electroporation causes a loss of control over ion channels, disrupting the balance of ions such as Na⁺ and K⁺, which interferes with normal neural signal transmission (causing immediate paralysis) [36]. Second, it induces a massive influx of extracellular Ca²⁺ (i.e., “calcium overload”) and triggers cellular “oxidative stress” [37,38]. The significant decline in AChE activity observed in Section 3.4 is the biochemical consequence of these cascades. We hypothesize that the inactivation occurs via three pathways:

Enzyme Degradation (Indirect Pathway): “Calcium overload” activates calcium-dependent proteases (e.g., Calpains), which initiate the degradation of intracellular proteins, including AChE.

Enzyme Oxidation (Indirect Pathway): Abundant reactive oxygen species generated by “oxidative stress” attack AChE proteins, oxidizing key amino acid residues. This alters the three-dimensional structure (denaturation), leading to inactivation.

Direct Inactivation (Direct Pathway): Furthermore, under the amplified high field strength caused by the “tip effect,” the strong electric field may directly act on AChE proteins. The field could disrupt the hydrogen bonds or salt bridges maintaining the tertiary structure via polarization or electro-mechanical forces, causing irreversible structural changes and loss of catalytic ability.

4.

Nervous System Dysfunction. The inactivation, degradation, or denaturation of AChE results in the failure to hydrolyze the neurotransmitter acetylcholine in the synaptic cleft. This leads to excessive accumulation of acetylcholine, causing continuous excitatory discharge of neurons. Ultimately, this results in nervous system dysfunction, muscle spasms, paralysis, and the death of the insect.

5.

Comparison with Chemical Control Mechanisms. This pathway shares similarities with the mechanism of organophosphorus insecticides, as both target AChE, but their triggering modes are fundamentally different [39,40]. Chemical insecticides inhibit enzymes via covalent binding of small molecules. In contrast, PEF operates through an “electric field-neurotoxicity” coupled pathway: the physical field causes enzymatic damage either directly or indirectly through electroporation-induced biochemical cascades. This reveals that PEF, as a physical insecticidal technology, operates on a more direct principle without chemical residue risks, providing theoretical support for its application as a green and sustainable strategy for stored grain pest control.

4.2. Application Potential: Penetration, Uniformity, and Quality Preservation

Beyond the lethal mechanism, the practical application potential of PEF in stored grain protection relies on its ability to treat bulk grain effectively without compromising quality.

1.

Penetration and Uniformity: A major challenge for physical disinfestation methods like cold plasma is limited penetration depth [12]. In contrast, our study demonstrates that PEF possesses excellent macroscopic penetration capabilities. The simulation results (Figure 9a,b) visually confirm that the electric field lines effectively penetrate the air voids between rice grains, maintaining sufficient field strength throughout the treatment chamber. This phenomenon is theoretically governed by the dielectric properties of the grain at the selected frequency (20 kHz). Unlike high-frequency electromagnetic waves (e.g., microwaves), where penetration is limited by surface absorption, dry rice behaves as a low-loss dielectric in the low-frequency, quasi-static electric field used in this study [28]. Consequently, the grain matrix does not effectively shield the electric field, allowing field lines to penetrate the depth of the processing layer with negligible attenuation. This theoretical finding is strongly supported by our “in-rice” validation experiment (Section 3.1.4), where a 100% mortality rate was achieved for T. castaneum mixed within the rice mass. This indicates that the heterogeneous nature of the grain pile does not significantly shield the insects from the electric field, ensuring a relatively uniform and effective treatment for hidden pests.

2.

Impact on Grain Quality: Preserving the nutritional and functional quality of grain is paramount. Unlike thermal methods such as radio frequency (RF) and microwave (MW), which rely on dielectric heating and often lead to the degradation of heat-sensitive nutrients due to excessive or non-uniform temperature rise [13], PEF operates fundamentally as a non-thermal process. Our thermal evaluation (Section 3.3) showed that the temperature rise during the optimal PEF treatment was negligible (< 1.5 °C). This “cold” processing characteristic suggests that PEF treatment is unlikely to cause thermal damage to the rice, thereby preserving its original quality, including heat-sensitive vitamins and proteins.

3.

Limitations and Future Perspectives: It should be noted that this study primarily focused on the adult stage of T. castaneum. Adults were selected because they are typically considered to have higher electrical resistance compared to eggs or larvae due to their well-developed, sclerotized exoskeleton, making them a robust model for optimizing lethal parameters [28]. However, the efficacy of PEF may vary across developmental stages due to differences in size, morphology, and dielectric properties. Therefore, while our results establish a solid baseline for adult control, future research will aim to systematically evaluate the lethal effects of PEF on eggs, larvae, and pupae to develop a comprehensive, multi-stage control strategy. Furthermore, the residue-free nature of PEF aligns strongly with growing global regulatory trends favoring the phase-out of traditional chemical fumigants and the adoption of sustainable processing technologies.

5. Conclusions

This study systematically optimized the process parameters for PEF extermination of Tribolium castaneum (T. castaneum) and, through physical simulation and experimental research, deeply explored its lethal mechanism. The main conclusions are as follows:

The electric field strength, pulse frequency, and treatment time of the PEF are all key factors affecting the mortality rate of T. castaneum, with electric field strength having the most significant effect. A predictive mortality model was successfully constructed based on response surface methodology (RSM), and the optimal process parameters were obtained: electric field strength of 26 kV/cm, frequency of 20 kHz, and treatment time of 140 s.

Under the optimal process parameters, the validation experiments achieved a 100% mortality rate, and a 100% extermination rate was also achieved in a simulated stored-grain environment (mixed with rice). This confirms the accuracy and reliability of the established model and demonstrates that PEF technology has great potential to penetrate grain gaps and be applied to practical stored-product pest control.

The electric field simulation results intuitively revealed the physical mechanism of action of PEF: due to the difference in permittivity, the T. castaneum body creates a “focusing effect” on the external electric field, causing the local electric field strength in nerve-ending-rich areas, such as the head and tail, to be much higher than the applied average field strength. This finding provides strong physical support for explaining the high efficiency of PEF insect control.

Mechanism studies indicate that the lethal effect of PEF is related to an “electro-neurotoxicity” mechanism. Crucially, the observed effects were confirmed to be primarily non-thermal, as dedicated evaluations showed negligible temperature increases during treatment. PEF treatment can significantly inhibit the activity of acetylcholinesterase (AChE) in T. castaneum, and the inhibitory effect is dose-dependent. Combined with the simulation results, the lethal pathway is deduced as: the “focusing effect” of the electric field induces “cell electroporation” in nerve cell membranes; electroporation acts as a physical trigger, further initiating biochemical cascades including calcium overload and oxidative stress, possibly supplemented by the direct action of the electric field, collectively leading to the structural damage and inactivation of AChE, which results in nervous system dysfunction and ultimately, pest death.

This research provides a solid theoretical basis from the perspectives of process optimization, physical field analysis, and biochemical mechanisms for PEF as a green, residue-free physical insect control technology. Compared with chemical fumigation (which typically requires 3–14 days), PEF significantly shortens the treatment time to 140 s without leaving residues. Unlike thermal treatments (e.g., radio frequency/microwave, RF/MW) that often raise grain temperature above 50 °C, PEF maintains a negligible temperature rise of <1.5 °C, effectively avoiding thermal damage to grain quality. Furthermore, unlike cold plasma (CP), which is typically restricted to surface treatment, PEF demonstrates superior macroscopic penetration suitable for bulk grain processing. Thus, PEF represents a rapid, residue-free, and quality-preserving alternative for sustainable stored-grain pest management.